Anti-fgfr3 antibodies and methods using same

ABSTRACT

The invention provides FGFR3 antibodies, and compositions comprising and methods of using these antibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent Ser. No. 15/356,483,filed Nov. 18, 2016, which is a continuation of U.S. patent applicationSer. No. 14/887,042, filed Oct. 19, 2015, issuing as U.S. Pat. No.9,499,623 on Nov. 22, 2016, which is a divisional of U.S. patentapplication Ser. No. 13/762,252, filed Feb. 7, 2013, issued as U.S. Pat.No. 9,161,977 on Oct. 20, 2015, which is a divisional of U.S. patentapplication Ser. No. 12/731,100 filed Mar. 24, 2010, issued as U.S. Pat.No. 8,410,250 on Apr. 2, 2013, and claims priority to U.S. ProvisionalApplication No. 61/163,222 filed on Mar. 25, 2009, the disclosures ofwhich are both incorporated by reference herein in their entirety,including drawings and sequence listings.

SEQUENCE LISTING

This application contains a Sequence Listing which has been submittedvia EFS-Web and is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to the field of molecularbiology. More specifically, the invention concerns anti-FGFR3antibodies, and uses of same.

BACKGROUND OF THE INVENTION

Fibroblast growth factors (FGFs) and their receptors (FGFRs) playcritical roles during embryonic development, tissue homeostasis andmetabolism (1-3). In humans, there are 22 FGFs (FGF1-14, FGF16-23) andfour FGF receptors with tyrosine kinase domain (FGFR1-4). FGFRs consistof an extracellular ligand binding region, with two or threeimmunoglobulin-like domains (IgD1-3), a single-pass transmembraneregion, and a cytoplasmic, split tyrosine kinase domain. FGFR1, 2 and 3each have two major alternatively spliced isoforms, designated IIIb andIIIc. These isoforms differ by about 50 amino acids in the second halfof IgD3, and have distinct tissue distribution and ligand specificity.In general, the IIIb isoform is found in epithelial cells, whereas IIIcis expressed in mesenchymal cells. Upon binding FGF in concert withheparan sulfate proteoglycans, FGFRs dimerize and become phosphorylatedat specific tyrosine residues. This facilitates the recruitment ofcritical adaptor proteins, such as FGFR substrate 2 α (FRS2α), leadingto activation of multiple signaling cascades, including themitogen-activated protein kinase (MAPK) and PI3K-AKT pathways (1, 3, 4).Consequently, FGFs and their cognate receptors regulate a broad array ofcellular processes, including proliferation, differentiation, migrationand survival, in a context-dependent manner.

Aberrantly activated FGFRs have been implicated in specific humanmalignancies (1, 5). In particular, the t(4;14) (p16.3;q32) chromosomaltranslocation occurs in about 15-20% of multiple myeloma patients,leading to overexpression of FGFR3 and correlates with shorter overallsurvival (6-9). FGFR3 is implicated also in conferring chemoresistanceto myeloma cell lines in culture (10), consistent with the poor clinicalresponse of t(4;14)+ patients to conventional chemotherapy (8).Overexpression of mutationally activated FGFR3 is sufficient to induceoncogenic transformation in hematopoietic cells and fibroblasts (11-14,15), transgenic mouse models (16), and murine bone marrowtransplantation models (16, 17). Accordingly, FGFR3 has been proposed asa potential therapeutic target in multiple myeloma. Indeed, severalsmall-molecule inhibitors targeting FGFRs, although not selective forFGFR3 and having cross-inhibitory activity toward certain other kinases,have demonstrated cytotoxicity against FGFR3-positive myeloma cells inculture and in mouse models (18-22).

FGFR3 overexpression has been documented also in a high fraction ofbladder cancers (23, 24). Furthermore, somatic activating mutations inFGFR3 have been identified in 60-70% of papillary and 16-20% ofmuscle-invasive bladder carcinomas (24, 25). In cell cultureexperiments, RNA interference (11, 26) or an FGFR3 single-chain Fvantibody fragment inhibited bladder cancer cell proliferation (27). Arecent study demonstrated that an FGFR3 antibody-toxin conjugateattenuates xenograft growth of a bladder cancer cell line throughFGFR3-mediated toxin delivery into tumors (28). However, it remainsunclear whether FGFR3 signaling is indeed an oncogenic driver of in vivogrowth of bladder tumors. Moreover, the therapeutic potential fortargeting FGFR3 in bladder cancer has not been defined on the basis ofin vivo models. Publications relating to FGFR3 and anti-FGFR3 antibodiesinclude U.S. Patent Publication no. 2005/0147612; Rauchenberger et al, JBiol Chem 278 (40):38194-38205 (2003); WO2006/048877;Martinez-Torrecuadrada et al, (2008) Mol Cancer Ther 7(4): 862-873;WO2007/144893; Trudel et al. (2006) 107(10): 4039-4046;Martinez-Torrecuadrada et al (2005) Clin Cancer Res 11 (17): 6280-6290;Gomez-Roman et al (2005) Clin Cancer Res 11:459-465; Direnzo, R et al(2007) Proceedings of AACR Annual Meeting, Abstract No. 2080;WO2010/002862. Crystal structures of FGFR3:anti-FGFR3 antibody aredisclosed in co-pending, co-owned U.S. patent application Ser. No.13/572,557 filed Aug. 10, 2012 as a continuation of U.S. patentapplication Ser. No. 12/661,852, filed Mar. 24, 2010.

It is clear that there continues to be a need for agents that haveclinical attributes that are optimal for development as therapeuticagents. The invention described herein meets this need and providesother benefits.

All references cited herein, including patent applications andpublications, are incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

The invention is based in part on the identification of a variety ofFGFR3 binding agents (such as antibodies, and fragments thereof). FGFR3presents an important and advantageous therapeutic target, and theinvention provides compositions and methods based on binding of theagents to FGFR3. FGFR3 binding agents of the invention, as describedherein, provide important therapeutic and diagnostic agents for use intargeting pathological conditions associated with expression and/oractivity of the FGFR3 signaling pathways. Accordingly, the inventionprovides methods, compositions, kits, and articles of manufacturerelated to FGFR3 binding.

The present invention provides antibodies that bind to FGFR3. In oneaspect, the invention features an isolated antibody that binds an FGFR3.In some embodiments, the antibody binds a FGFR3 IIIb isoform and/or aFGFR3 IIIc isoform. In some embodiments, the antibody binds a mutatedFGFR3 (e.g., one or more of FGFR3 IIIb R248C, S249C, G372C, Y375C,K652E, and/or one or more of FGFR3 IIIc R248C, S249C, G370C, Y373C,K650E). In some embodiments, the antibody binds monomeric FGFR3 (e.g.,monomeric FGFR3 IIIb and/or IIIc isoforms). In some embodiments, theantibody promotes formation of monomeric FGFR3, such as by stabilizingthe monomeric FGFR3 form relative to the dimeric FGFR3 form.

In one aspect, the invention provides an isolated anti-FGFR3 antibody,wherein a full length IgG form of the antibody binds human FGFR3 with aKd of 1×10⁻⁷ or stronger. As is well-established in the art, bindingaffinity of a ligand to its receptor can be determined using any of avariety of assays, and expressed in terms of a variety of quantitativevalues. Accordingly, in one embodiment, the binding affinity isexpressed as Kd values and reflects intrinsic binding affinity (e.g.,with minimized avidity effects). Generally and preferably, bindingaffinity is measured in vitro, whether in a cell-free or cell-associatedsetting. Any of a number of assays known in the art, including thosedescribed herein, can be used to obtain binding affinity measurements,including, for example, Biacore, radioimmunoassay (RIA), and ELISA. Insome embodiments, the full length IgG form of the antibody binds humanFGFR3 with a Kd of 1×10⁻⁸ or stronger, with a Kd of 1×10⁻⁹ or stronger,or with a Kd of 1×10⁻¹⁰ or stronger.

Generally, the anti-FGFR3 antibodies of the present invention areantagonist antibodies. Thus, in one aspect, the anti-FGFR3 antibodiesinhibit FGFR3 activity (e.g., FGFR3-IIIb and/or FGFR3-IIIc activity). Insome embodiments, the anti-FGFR3 antibody (generally in bivalent form)does not possess substantial FGFR3 agonist function. In someembodiments, the anti-FGFR3 antagonist antibody (generally in bivalentform) possesses little or no FGFR3 agonist function. In one embodiment,an antibody of the invention (generally in bivalent form) does notexhibit an FGFR3 agonist activity level that is above background levelthat is of statistical significance.

In one aspect, binding of the antibody to a FGFR3 may inhibitdimerization of the receptor with another unit of the receptor, wherebyactivation of the receptor is inhibited (due, at least in part, to alack of receptor dimerization). Inhibition can be direct or indirect.

In one aspect, the invention provides anti-FGFR3 antibodies that do notpossess substantial apoptotic activity (e.g., does not induce apoptosisof a cell, e.g., a transitional cell carcinoma cell or a multiplemyeloma cell, such as a multiple myeloma cell comprising a FGFR3translocation, such as a t(4; 14) translocation). In some embodiments,the anti-FGFR3 antibody possesses little or no apoptotic function. Insome embodiment, the FGFR3 antibodies do not exhibit apoptotic functionthat is above background level that is of statistical significance.

In one aspect, the invention provides anti-FGFR3 antibodies that do notinduce substantial FGFR3 down-regulation. In some embodiments, theanti-FGFR3 antibody induces little or no receptor down-regulation. Insome embodiment, the FGFR3 antibodies do not induce receptordown-regulation that is above background level that is of statisticalsignificance.

In one aspect, the invention provides anti-FGFR3 antibodies that possesseffector function. In one embodiment, the effector function comprisesantibody-dependent cell-mediated cytotoxicity (ADCC). In one embodiment,the anti-FGFR3 antibody (in some embodiments, a naked anti-FGFR3antibody) is capable of killing a cell, in some embodiments, a multiplemyeloma cells (e.g., multiple myeloma cells comprising a translocation,e.g., a t(4; 14) translocation). In some embodiments, the anti-FGFR3antibody is capable of killing a cell that expresses about 10,000 FGFR3molecules per cell or more (such as about 11,000, about 12,000, about13,000, about 14,000, about 15,000, about 16,000, about 17,000, about18,000 or more FGFR3 molecules per cell). In other embodiments, the cellexpresses about 2000, about 3000, about 4000, about 5000, about 6000,about 7000, about 8000, or more FGFR3 molecules per cell.

In one aspect, the anti-FGFR3 antibody of the invention inhibitsconstitutive FGFR3 activity. In some embodiments, constitutive FGFR3activity is ligand-dependent FGFR3 constitutive activity. In someembodiments, constitutive FGFR3 activity is ligand-independentconstitutive FGFR3 activity.

In one aspect, the anti-FGFR3 antibody inhibits FGFR3 comprising amutation corresponding to FGFR3-IIIb^(R248C). As used herein the term“comprising a mutation corresponding to FGFR3-IIIb^(R248C)” isunderstood to encompass FGFR3-IIIb^(R248C) and FGFR3-IIIc^(R248C), aswell as additional FGFR3 forms comprising an R to C mutation at aposition corresponding to FGFR3-IIIb R248. One of ordinary skill in theart understands how to align FGFR3 sequences in order identifycorresponding residues between respective FGFR3 sequences, e.g.,aligning a FGFR3-IIIc sequence with a FGFR3-IIIb sequence to identifythe position in FGFR3 corresponding R248 position in FGFR3-IIIb. In someembodiments, the anti-FGFR3 antibody inhibits FGFR3-IIIb^(R248C) and/orFGFR3-IIIc^(R248C)In one aspect, the anti-FGFR3 antibodies inhibit FGFR3comprising a mutation corresponding to FGFR3-IIIb^(K652E). Forconvenience, the term “comprising a mutation corresponding toFGFR3-IIIb^(K652E)” is understood to encompass FGFR3-IIIb^(K652E) andFGFR3-IIIc^(K650E), as well as additional FGFR3 forms comprising an K toE mutation at a position corresponding to FGFR3-IIIb K652. One ofordinary skill in the art understands how to align FGFR3 sequences inorder identify corresponding residues between respective FGFR3sequences, e.g., aligning a FGFR3-IIIc sequence with a FGFR3-IIIbsequence to identify the position in FGFR3 corresponding K652 positionin FGFR3-IIIb. In some embodiments, the anti-FGFR3 antibody inhibitsFGFR3-IIIb^(K652E) and/or FGFR3-IIIc^(K650E).

In one aspect, the anti-FGFR3 antibodies inhibit FGFR3 comprising amutation corresponding to FGFR3-IIIb^(S249C). For convenience, the term“comprising a mutation corresponding to FGFR3-IIIb^(S249C)” isunderstood to encompass FGFR3-IIIb^(S249C) and FGFR3-IIIc^(S249C), aswell as additional FGFR3 forms comprising an S to C mutation at aposition corresponding to FGFR3-IIIb S249. In some embodiments, theanti-FGFR3 antibody inhibits FGFR3-IIIb^(S249C) and/orFGFR3-IIIc^(S249C).

In one aspect, the anti-FGFR3 antibodies inhibit FGFR3 comprising amutation corresponding to FGFR3-IIIb^(G372C). For convenience, the term“comprising a mutation corresponding to FGFR3-IIIb^(G372C)” isunderstood to encompass FGFR3-IIIb^(G372C) and FGFR3-IIIc^(G370C), aswell as additional FGFR3 forms comprising a G to C mutation at aposition corresponding to FGFR3-IIIb G372. In some embodiments, theanti-FGFR3 antibody inhibits FGFR3-IIIb^(G372C) and/orFGFR3-IIIc^(G370C).

In one aspect, the anti-FGFR3 antibodies inhibit FGFR3 comprising amutation corresponding to FGFR3-IIIb^(Y375C). For convenience, the term“comprising a mutation corresponding to FGFR3-IIIb^(Y375C)” isunderstood to encompass FGFR3-IIIb^(Y375C) and FGFR3-IIIc^(Y373C), aswell as additional FGFR3 forms comprising an S to C mutation at aposition corresponding to FGFR3-IIIb S249. In some embodiments, theanti-FGFR3 antibody inhibits FGFR3-IIIb^(Y375C) and/orFGFR3-IIIc^(Y373C).

In one aspect, the anti-FGFR3 antibodies inhibit (a) FGFR3-IIIb^(K652E)and (b) one or more of FGFR3-IIIb^(R248C), FGFR3-IIIb^(Y375C),FGFR3-IIIb^(S249C), and FGFR3IIIb^(G372C).

In one aspect, the anti-FGFR3 antibodies inhibit (a) FGFR3-IIIc^(K650E)and (b) one or more of FGFR3-IIIc^(R248C), FGFR3-IIIc^(Y373C),FGFR3-IIIc^(S249C), and FGFR3IIIc^(G370C).

In one aspect, the anti-FGFR3 antibodies inhibit (a) FGFR3-IIIb^(R248C)and (b) one or more of FGFR3-IIIb^(K652E), FGFR3-IIIb^(Y375C),FGFR3-IIIb^(S249C), and FGFR3-IIIb^(G372C).

In one aspect, the anti-FGFR3 antibodies inhibit (a) FGFR3-IIIc^(R248C)and (b) one or more of FGFR3-IIIc^(K650E), FGFR3-IIIc^(Y373C),FGFR3-IIIc^(S249C), and FGFR3-IIIc^(G370C).

In one aspect, the anti-FGFR3 antibodies inhibit (a) FGFR3-IIIb^(G372C)and (b) one or more of FGFR3-IIIb^(K652E), FGFR3-IIIb^(Y375C),FGFR3-IIIb^(S249C), and FGFR3-IIIb^(R248C).

In one aspect, the anti-FGFR3 antibodies inhibit (a) FGFR3-IIIc^(G370C)and (b) one or more of FGFR3-IIIc^(K650E), FGFR3-IIIc^(Y373C),FGFR3-IIIc^(S249C), and FGFR3-IIIc^(R248C).

In one aspect, the anti-FGFR3 antibodies inhibit FGFR3-IIIb^(R248C),FGFR3-IIIb^(K652E), FGFR3-IIIb^(Y375C), FGFR3-IIIb^(S249C), andFGFR3-IIIb^(G372C).

In one aspect, the anti-FGFR3 antibodies inhibit FGFR3-IIIc^(R248C),FGFR3-IIIc^(K650E), FGFR3-IIIc^(Y373C), FGFR3-IIIc^(S249C), andFGFR3-IIIc^(G370C).

In one aspect, the invention provides an isolated anti-FGFR3 antibodycomprising:

(a) at least one, two, three, four, or five hypervariable region (HVR)sequences selected from:

(i) HVR-L1 comprising sequence A1-A11, wherein A1-A11 is RASQDVDTSLA(SEQ ID NO:87),

(ii) HVR-L2 comprising sequence B1-B7, wherein B1-B7 is SASFLYS (SEQ IDNO:88),

(iii) HVR-L3 comprising sequence C1-C9, wherein C1-C9 is QQSTGHPQT (SEQID NO:89),

(iv) HVR-H1 comprising sequence D1-D10, wherein D1-D10 is GFTFTSTGIS(SEQ ID NO:84),

(v) HVR-H2 comprising sequence E1-E18, wherein E1-E18 isGRIYPTSGSTNYADSVKG (SEQ ID NO:85), and

(vi) HVR-H3 comprising sequence F1-F20, wherein F1-F20 isARTYGIYDLYVDYTEYVMDY (SEQ ID NO:86); and

(b) at least one variant HVR, where the variant HVR sequence comprisesmodification of at least one residue (at least two residues, at leastthree or more residues) of the sequence depicted in SEQ ID NOS:1-18,48-131 and 140-145. The modification desirably is a substitution,insertion, or deletion.

In some embodiments, a HVR-L1 variant comprises 1-6 (1, 2, 3, 4, 5, or6) substitutions in any combination of the following positions: A5 (V orD), A6 (V or I), A7 (D, E or S), A8 (T or I), A9 (A or S) and A10 (V orL). In some embodiments, a HVR-L2 variant comprises 1-2 (1 or 2)substitutions in any combination of the following positions: B1 (S orG), B4 (F or S or T) and B6 (A or Y). In some embodiments, a HVR-L3variant comprises 1-6 (1, 2, 3, 4, 5, or 6) substitutions in anycombination of the following positions: C3 (G or S or T), C4 (T or Y orA), C5 (G or S or T or A), C6 (A or H or D or T or N), C7 (Q or P or S),and C8 (S or Y or L or P or Q). In some embodiment, a HVR-H1 variantcomprises 1-3 (1, 2, or 3) substitutions in any combination of thefollowing positions: D3 (S or T), D5 (W or Y or S or T), D6 (S or G orT). In some embodiment, a HVR-H2 variant comprises 1-6 (1, 2, 3, 4, 5,or 6) substitutions in any combination of the following positions: E2 (Ror S), E6 (Y or A or L or S or T), E7 (A or Q or D or G or Y or S or Nor F), E8 (A or D or G), E9 (T or S), E10 (K or F or T or S), E11 (Y orH or N or I).

In one aspect, the invention provides an isolated anti-FGFR3 antibodycomprising:

(a) at least one, two, three, four, or five hypervariable region (HVR)sequences selected from:

(i) HVR-L1 comprising sequence RASQX₁X₂X₃X₄X₅ X₆A, wherein X₁ is V or D,X₂ is V or I, X₃ is D, E or S, X₄ is T or I, X₅ is A or S, and X₆ is Vor L (SEQ ID NO: 146),

(ii) HVR-L2 comprising sequence X₁ASFLX₂S wherein X₁ is S or G and X₂ isA or Y (SEQ ID NO: 147),

(iii) HVR-L3 comprising sequence QQX₁X₂X₃X₄X₅X₆T, wherein X₁ is G, S orT, X₂ is T, Y or A, X₃ is G, S, T, or A, X₄ is A, H, D, T, or N, X₅ isQ, P or S, X₆ is S, Y, L, P or Q (SEQ ID NO: 148),

(iv) HVR-H1 comprising sequence GFX₁FX₂X₃TGIS, wherein X₁ is S or T, X₂is W, Y, S or T, X₃ is S, G, or T (SEQ ID NO: 149),

(v) HVR-H2 comprising sequence GRIYPX₁X₂X₃X₄X₅X₆YADSVKG, wherein X₁ isY, A, L, S, or T, X₂ is A, Q, D, G, Y, S, N or F, X₃ is A, D, or G, X₄is T or S, X₅ is K, F, T, or S, X₆ is Y, H, N or I (SEQ ID NO:150), and

(vi) HVR-H3 comprising sequence ARTYGIYDLYVDYTEYVMDY (SEQ ID NO: 151).

In some embodiments, HVR-L1 comprises sequence RASQXIVX₂X₃X₄VA, whereinX₁ is V or D, X₂ is D, E or S, X₃ is T or I, X₄ is A or S (SEQ IDNO:152). In some embodiments, HVR-L3 comprises sequence QQX₁X₂X₃X₄X₅X₆T,wherein X₁ is S, G, or T, X₂ is Y, T, or A, X₃ is T or G, X₄ is T, H orN, X₅ is P or S, X₆ is P, Q, Y, or L (SEQ ID NO: 153). In someembodiments, HVR-H2 comprises sequence GRIYPX₁X₂GSTX₃YADSVKG, wherein X₁is T or L, X₂ is N, Y, S, G, A, or Q; X₃ is N or H (SEQ ID NO: 154).

In another aspect, the invention features an isolated anti-FGFR3antibody that comprises one, two, three, four, five, or six HVRs, whereeach HVR comprises, consists, or consists essentially of a sequenceselected from SEQ ID NOS:1-18, 48-131 and 140-145, and where SEQ IDNO:1, 7, 13, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126or 143 corresponds to an HVR-H1, SEQ ID NO:2, 8, 14, 49, 55, 61, 67, 73,79, 85, 91, 97, 103, 109, 115, 121, 127 or 144 corresponds to an HVR-H2,SEQ ID NO:3, 9, 15, 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 110, 116,122, 128 or 145 corresponds to an HVR-H3, SEQ ID NO:4, 10, 16, 51, 57,63, 69, 75, 81, 87, 93, 99, 105, 111, 117, 123, 129 or 140 correspondsto an HVR-L1, SEQ ID NO:5, 11, 17, 52, 58, 64, 70, 76, 82, 88, 94, 100,106, 112, 118, 124, 130 or 141 corresponds to an HVR-L2, and SEQ IDNO:6, 12, 18, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113, 119, 125,131 or 142 corresponds to an HVR-L3.

In one aspect, the invention provides an anti-FGFR3 antibody comprisinga HVR-H1 comprising the sequence of SEQ ID NO: 1, 7, 13, 48, 54, 60, 66,72, 78, 84, 90, 96, 102, 108, 114, 120, 126 or 143.

In one aspect, the invention provides an anti-FGFR3 antibody comprisinga HVR-H2 comprising the sequence of SEQ ID NO:2, 8, 14, 49, 55, 61, 67,73, 79, 85, 91, 97, 103, 109, 115, 121, 127 or 144.

In one aspect, the invention provides an anti-FGFR3 antibody comprisinga HVR-H3 comprising the sequence of SEQ ID NO:3, 9, 15, 50, 56, 62, 68,74, 80, 86, 92, 98, 104, 110, 116, 122, 128 or 145.

In one aspect, the invention provides an anti-FGFR3 antibody comprisinga HVR-L1 region comprising the sequence of SEQ ID NO:4, 10, 16, 51, 57,63, 69, 75, 81, 87, 93, 99, 105, 111, 117, 123, 129 or 140.

In one aspect, the invention provides an anti-FGFR3 antibody comprisinga HVR-L2 region comprising the sequence of SEQ ID NO:5, 11, 17, 52, 58,64, 70, 76, 82, 88, 94, 100, 106, 112, 118, 124, 130 or 141.

In one aspect, the invention provides an anti-FGFR3 antibody comprisinga HVR-L3 region comprising the sequence of SEQ ID NO:6, 12, 18, 53, 59,65, 71, 77, 83, 89, 95, 101, 107, 113, 119, 125, 131 or 142.

In one aspect, an anti-FGFR3 antibody comprises a heavy chain variableregion comprising HVR-H1, HVR-H2, HVR-H3, wherein each, in order,comprises SEQ ID NO: 1, 2, 3, and/or a light chain variable regioncomprising HVR-L1, HVR-L2, and HVR-L3, where each, in order, containsSEQ ID NO: 4, 5, 6.

In another aspect, an-anti-FGFR3 antibody comprises a heavy chainvariable region comprising HVR-H1, HVR-H2, HVR-H3, wherein each, inorder, comprises SEQ ID NO:7, 8, 9, and/or a light chain variable regioncomprising HVR-L1, HVR-L2, and HVR-L3, where each, in order, comprisesSEQ ID NO: 10, 11, 12.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising HVR-H1, HVR-H2, HVR-H3, where each, in order,comprises SEQ ID NO: 13, 14, 15, and/or a light chain variable regioncomprising HVR-L1, HVR-L2, and HVR-L3, where each, in order, comprisesSEQ ID NO:16, 17, 18.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising HVR-H1, HVR-H2, HVR-H3, where each, in order,comprises SEQ ID NO: 48, 49, 50, and/or a light chain variable regionHVR-L1, HVR-L2, and HVR-L3, where each, in order, comprises SEQ ID NO:51, 52, 53.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising HVR-H1, HVR-H2, HVR-H3, where each, in order,comprises SEQ ID NO: 54, 55, 56, and/or a light chain variable regioncomprising HVR-L1, HVR-L2, and HVR-L3, where each, in order, comprisesSEQ ID NO: 57, 58, 59.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising HVR-H1, HVR-H2, HVR-H3, where each, in order,comprises SEQ ID NO:60, 61, 62, 63, and/or a light chain variable regioncomprising HVR-L1, HVR-L2, and HVR-L3, where each, in order, comprisesSEQ ID NO: 63, 64, 65.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising HVR-H1, HVR-H2, HVR-H3, where each, in order,comprises SEQ ID NO:66, 67, 68, and/or a light chain variable regioncomprising HVR-L1, HVR-L2, and HVR-L3, where each, in order, comprisesSEQ ID NO: 69, 70, 71.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising HVR-H1, HVR-H2, HVR-H3, where each, in order,comprises SEQ ID NO:72, 73, 74, and/or a light chain variable regioncomprising HVR-L1, HVR-L2, and HVR-L3, where each, in order, comprisesSEQ ID NO: 75, 76, 77.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising HVR-H1, HVR-H2, HVR-H3, where each, in order,comprises SEQ ID NO:78, 79 80, and/or a light chain variable regioncomprising HVR-L1, HVR-L2, and HVR-L3, where each, in order, comprisesSEQ ID NO:81, 82, 83.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising HVR-H1, HVR-H2, HVR-H3, where each, in order,comprises SEQ ID NO: 84, 85, 86, and/or a light chain variable regioncomprising HVR-L1, HVR-L2, and HVR-L3, where each, in order, comprisesSEQ ID NO:87, 88, 89.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising HVR-H1, HVR-H2, HVR-H3, where each, in order,comprises SEQ ID NO: 90, 91, 92, and/or a light chain variable regioncomprising HVR-L1, HVR-L2, and HVR-L3, where each, in order, comprisesSEQ ID NO:93, 94, 95.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising HVR-H1, HVR-H2, HVR-H3, where each, in order,comprises SEQ ID NO: 96, 97, 98, and/or a light chain variable regioncomprising HVR-L1, HVR-L2, and HVR-L3, where each, in order, comprisesSEQ ID NO: 99, 100, 101.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising HVR-H1, HVR-H2, HVR-H3, where each, in order,comprises SEQ ID NO: 102, 103, 104, and/or a light chain variable regioncomprising HVR-L1, HVR-L2, and HVR-L3, where each, in order, comprisesSEQ ID NO: 105, 106, 107.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising HVR-H1, HVR-H2, HVR-H3, where each, in order,comprises SEQ ID NO: 108, 109, 110, and/or a light chain variable regioncomprising HVR-L1, HVR-L2, and HVR-L3, where each, in order, comprisesSEQ ID NO: 111, 112, 113.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising HVR-H1, HVR-H2, HVR-H3, where each, in order,comprises SEQ ID NO: 114, 115, 116, and/or a light chain variable regioncomprising HVR-L 1, HVR-L2, and HVR-L3, where each, in order, comprisesSEQ ID NO: 117, 118, 119.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising HVR-H1, HVR-H2, HVR-H3, where each, in order,comprises SEQ ID NO: 120, 121, 122, and/or a light chain variable regioncomprising HVR-L1, HVR-L2, and HVR-L3, where each, in order, comprisesSEQ ID NO: 123, 124, 125.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising HVR-H1, HVR-H2, HVR-H3, where each, in order,comprises SEQ ID NO: 126, 127, 128, and/or a light chain variable regioncomprising HVR-L1, HVR-L2, and HVR-L3, where each, in order, comprisesSEQ ID NO:129, 130, 131.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising HVR-H1, HVR-H2, HVR-H3, where each, in order,comprises SEQ ID NO: 143, 144, 145, and/or a light chain variable regioncomprising HVR-L1, HVR-L2, and HVR-L3, where each, in order, comprisesSEQ ID NO: 140, 141, 142.

The amino acid sequences of SEQ ID NOs:1-18, 48-131 and 140-145 arenumbered with respect to individual HVR (i.e., H1, H2 or H3) asindicated in FIGS. 1A, 1B and 1C, the numbering being consistent withthe Kabat numbering system as described below.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising SEQ ID NO:132 and a light chain variableregion.

In another aspect, an anti-FGFR3 antibody comprises a light chainvariable region comprising SEQ ID NO: 133, and a heavy chain variableregion.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising SEQ ID NO:132 and a light chain variableregion comprising SEQ ID NO:133.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising SEQ ID NO:134 and a light chain variableregion.

In another aspect, an anti-FGFR3 antibody comprises a light chainvariable region comprising SEQ ID NO: 135, and a heavy chain variableregion.

In another aspect, an anti-FGFR3 antibody comprises a light chainvariable region comprising SEQ ID NO: 139, and a heavy chain variableregion.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising SEQ ID NO: 134 and a light chain variableregion comprising SEQ ID NO: 135.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising SEQ ID NO: 136 and a light chain variableregion.

In another aspect, an anti-FGFR3 antibody comprises a light chainvariable region comprising SEQ ID NO: 137, and a heavy chain variableregion.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising SEQ ID NO: 136 and a light chain variableregion comprising SEQ ID NO: 137.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising SEQ ID NO: 138 and a light chain variableregion.

In another aspect, an anti-FGFR3 antibody comprises a light chainvariable region comprising SEQ ID NO: 139, and a heavy chain variableregion.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising SEQ ID NO: 138 and a light chain variableregion comprising SEQ ID NO: 139.

In one aspect, the invention provides an anti-FGFR3 antibody comprising:at least one, two, three, four, five, and/or six hypervariable region(HVR) sequences selected from the group consisting of:

(a) HVR-L1 comprising sequence (SEQ ID NO: 155) SASSSVSYMH,(SEQ ID NO: 156) SASSSVSYMH or (SEQ ID NO: 157) LASQTIGTWLA,(b) HVR-L2 comprising sequence (SEQ ID NO: 158) TWIYDTSILAS,(SEQ ID NO: 159) RWIYDTSKLAS, or (SEQ ID NO: 160) LLIYAATSLAD,(c) HVR-L3 comprising sequence (SEQ ID NO: 161) QQWTSNPLT,(SEQ ID NO: 162) QQWSSYPPT, or (SEQ ID NO: 163) QQLYSPPWT,(d) HVR-H1 comprising sequence (SEQ ID NO: 164) GYSFTDYNMY,(SEQ ID NO: 165) GYVFTHYNMY, or (SEQ ID NO: 166) GYAFTSYNMY,(e) HVR-H2 comprising sequence (SEQ ID NO: 167) WIGYIEPYNGGTSYNQKFKG,(SEQ ID NO: 168) WIGYIEPYNGGTSYNQKFKG, or (SEQ ID NO: 169)WIGYIDPYIGGTSYNQKFKG, and (f) HVR-H3 comprising sequence(SEQ ID NO: 170) ASPNYYDSSPFAY, (SEQ ID NO: 171) ARGQGPDFDV, or(SEQ ID NO: 172) ARWGDYDVGAMDY.

In one aspect, the invention provides an anti-FGFR3 antibody comprising:at least one, two, three, four, five, and/or six hypervariable region(HVR) sequences selected from the group consisting of:

(a) HVR-L1 comprising sequence (SEQ ID NO: 155) SASSSVSYMH,(b) HVR-L2 comprising sequence (SEQ ID NO: 158) TWIYDTSILAS,(c) HVR-L3 comprising sequence (SEQ ID NO: 161) QQWTSNPLT,(d) HVR-H1 comprising sequence (SEQ ID NO: 164) GYSFTDYNMY,(e) HVR-H2 comprising sequence (SEQ ID NO: 167) WIGYIEPYNGGTSYNQKFKG,and (f) HVR-H3 comprising sequence (SEQ ID NO: 170) ASPNYYDSSPFAY.

In one aspect, the invention provides an anti-FGFR3 antibody comprising:at least one, two, three, four, five, and/or six hypervariable region(HVR) sequences selected from the group consisting of:

(a) HVR-L1 comprising sequence (SEQ ID NO: 156) SASSSVSYMH,(b) HVR-L2 comprising sequence (SEQ ID NO: 159) RWIYDTSKLAS,(c) HVR-L3 comprising sequence (SEQ ID NO: 162) QQWSSYPPT,(d) HVR-H1 comprising sequence (SEQ ID NO: 165) GYVFTHYNMY,(e) HVR-H2 comprising sequence (SEQ ID NO: 168) WIGYIEPYNGGTSYNQKFKG,and (f) HVR-H3 comprising sequence (SEQ ID NO: 171) ARGQGPDFDV.

In one aspect, the invention provides an anti-FGFR3 antibody comprising:at least one, two, three, four, five, and/or six hypervariable region(HVR) sequences selected from the group consisting of:

(a) HVR-L1 comprising sequence (SEQ ID NO: 157) LASQTIGTWLA,(b) HVR-L2 comprising sequence (SEQ ID NO: 160) LLIYAATSLAD,(c) HVR-L3 comprising sequence (SEQ ID NO: 163) QQLYSPPWT,(d) HVR-H1 comprising sequence (SEQ ID NO: 166) GYAFTSYNMY,(e) HVR-H2 comprising sequence (SEQ ID NO: 169) WIGYIDPYIGGTSYNQKFKG,and (f) HVR-H3 comprising sequence (SEQ ID NO: 172) ARWGDYDVGAMDY.

In one aspect, the invention provides an anti-FGFR3 antibody comprising(a) a light chain comprising (i) HVR-L1 comprising sequence SASSSVSYMH(SEQ ID NO:155); (ii) HVR-L2 comprising sequence TWIYDTSILAS (SEQ ID NO:158); and (iii) HVR-L3 comprising sequence QQWTSNPLT (SEQ ID NO: 161);and/or (b) a heavy chain comprising (i) HVR-H1 comprising sequenceGYSFTDYNMY (SEQ ID NO: 164); (ii) HVR-H2 comprising sequenceWIGYIEPYNGGTSYNQKFKG (SEQ ID NO: 167); and (iii) HVR-H3 comprisingsequence ASPNYYDSSPFAY (SEQ ID NO: 170).

In one aspect, the invention provides an anti-FGFR3 antibody comprising(a) a light chain comprising (i) HVR-L1 comprising sequence SASSSVSYMH(SEQ ID NO:156); (ii) HVR-L2 comprising sequence RWIYDTSKLAS (SEQ ID NO:159); and (iii) HVR-L3 comprising sequence QQWSSYPPT (SEQ ID NO: 162);and/or (b) a heavy chain comprising (i) HVR-H1 comprising sequenceGYVFTHYNMY (SEQ ID NO: 165); (ii) HVR-H2 comprising sequenceWIGYIEPYNGGTSYNQKFKG (SEQ ID NO: 168); and (iii) HVR-H3 comprisingsequence ARGQGPDFDV (SEQ ID NO: 171).

In one aspect, the invention provides an anti-FGFR3 antibody comprising(a) a light chain comprising (i) HVR-L1 comprising sequence LASQTIGTWLA(SEQ ID NO: 157); (ii) HVR-L2 comprising sequence LLIYAATSLAD (SEQ IDNO: 160); and (iii) HVR-L3 comprising sequence QQLYSPPWT (SEQ ID NO:163); and/or (b) a heavy chain comprising (i) HVR-H1 comprising sequenceGYAFTSYNMY (SEQ ID NO: 166); (ii) HVR-H2 comprising sequenceWIGYIDPYIGGTSYNQKFKG (SEQ ID NO: 169); and (iii) HVR-H3 comprisingsequence ARWGDYDVGAMDY (SEQ ID NO:172). Some embodiments of antibodiesof the invention comprise a light chain variable domain of humanized 4D5antibody (huMAb4D5-8) (HERCEPTIN®, Genentech, Inc., South San Francisco,Calif., USA) (also referred to in U.S. Pat. No. 6,407,213 and Lee etal., J. Mol. Biol. (2004), 340(5):1073-1093) as depicted in SEQ IDNO:173 below.

(SEQ ID NO: 173)1 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly AspArg Val Thr Ile Thr Cys 

Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser AlaSer Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 

 Ser GlyThr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala ThrTyr Tyr Cys 

 Phe Gly Gln Gly ThrLys Val Glu Ile Lys 107 (HVR residues are underlined)In one embodiment, the huMAb4D5-8 light chain variable domain sequenceis modified at one or more of positions 30, 66, and 91 (Asn, Arg, andHis as indicated in bold/italics above, respectively). In a particularembodiment, the modified huMAb4D5-8 sequence comprises Ser in position30, Gly in position 66, and/or Ser in position 91. Accordingly, in oneembodiment, an antibody of the invention comprises a light chainvariable domain comprising the sequence depicted in SEQ ID NO:174 below:

(SEQ ID NO: 174)1 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly AspArg Val Thr Ile Thr Cys 

Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser AlaSer Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

 Ser GlyThr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala ThrTyr Tyr Cys 

 Phe Gly Gln Gly ThrLys Val Glu Ile Lys 107 (HVR residues are underlined)Substituted residues with respect to huMAb4D5-8 are indicated inbold/italics.

Antibodies of the invention can comprise any suitable framework variabledomain sequence, provided binding activity to FGFR3 is substantiallyretained. For example, in some embodiments, antibodies of the inventioncomprise a human subgroup III heavy chain framework consensus sequence.In one embodiment of these antibodies, the framework consensus sequencecomprises a substitution at position 71, 73, and/or 78. In someembodiments of these antibodies, position 71 is A, 73 is T and/or 78 isA. In one embodiment, these antibodies comprise heavy chain variabledomain framework sequences of huMAb4D5-8 (HERCEPTIN®, Genentech, Inc.,South San Francisco, Calif., USA) (also referred to in U.S. Pat. Nos.6,407,213 & 5,821,337, and Lee et al., J. Mol. Biol. (2004),340(5):1073-1093). In one embodiment, these antibodies further comprisea human κI light chain framework consensus sequence. In a particularembodiment, these antibodies comprise light chain HVR sequences ofhuMAb4D5-8 as described in U.S. Pat. Nos. 6,407,213 & 5,821,337.) In oneembodiment, these antibodies comprise light chain variable domainsequences of huMAb4D5-8 (HERCEPTIN®, Genentech, Inc., South SanFrancisco, Calif., USA) (also referred to in U.S. Pat. Nos. 6,407,213 &5,821,337, and Lee et al., J. Mol. Biol. (2004), 340(5):1073-1093).

In one embodiment, an antibody of the invention comprises a heavy chainvariable domain, wherein the framework sequence comprises the sequenceof SEQ ID NOS: 19 and 203-205, 20 and 206-208, 21 and 209-211, 22 and212-214, 23 and 215-217, 24 and 218-220, 25 and 221-223, 26 and 224-226,27 and 227-229, 28 and 230-232, 29 and 233-235, 30 and 236-238, 31 and239-241, 32 and 242-244, 33 and 245-247, 34 and 248-250, 35 and 251-253,36 and 254-256, and/or 37 and 257-259, and HVR H1, H2, and H3 sequencesare SEQ ID NOS: 13, 14 and/or 15, respectively. In another embodiment,the framework sequence comprises the sequence of SEQ ID NOS: 19 and203-205, 20 and 206-208, 21 and 209-211, 22 and 212-214, 23 and 215-217,24 and 218-220, 25 and 221-223, 26 and 224-226, 27 and 227-229, 28 and230-232, 29 and 233-235, 30 and 236-238, 31 and 239-241, 32 and 242-244,33 and 245-247, 34 and 248-250, 35 and 251-253, 36 and 254-256, and/or37 and 257-259, and HVR H1, H2, and H3 sequences are SEQ ID NOS:48, 49and/or 50, respectively. In yet another embodiment, the frameworksequence comprises the sequence of SEQ ID NOS: 19 and 203-205, 20 and206-208, 21 and 209-211, 22 and 212-214, 23 and 215-217, 24 and 218-220,25 and 221-223, 26 and 224-226, 27 and 227-229, 28 and 230-232, 29 and233-235, 30 and 236-238, 31 and 239-241, 32 and 242-244, 33 and 245-247,34 and 248-250, 35 and 251-253, 36 and 254-256, and/or 37 and 257-259,and HVR H1, H2, and H3 sequences are SEQ ID NOS:84, 85, and/or 86,respectively. In a further embodiment, the framework sequence comprisesthe sequence of SEQ ID NOS: 19 and 203-205, 20 and 206-208, 21 and209-211, 22 and 212-214, 23 and 215-217, 24 and 218-220, 25 and 221-223,26 and 224-226, 27 and 227-229, 28 and 230-232, 29 and 233-235, 30 and236-238, 31 and 239-241, 32 and 242-244, 33 and 245-247, 34 and 248-250,35 and 251-253, 36 and 254-256, and/or 37 and 257-259, and HVR H1, H2,and H3 sequences are SEQ ID NOS: 108, 109, and/or 110, respectively.

In a particular embodiment, an antibody of the invention comprises alight chain variable domain, wherein the framework sequence comprisesthe sequence of SEQ ID NOS:38 and 260-262, 39 and 263-265, 40 and266-268, and/or 41 and 269-271, and HVR L1, L2, and L3 sequences are SEQID NOS:16, 17, and/or 18, respectively. In another embodiment, anantibody of the invention comprises a light chain variable domain,wherein the framework sequence comprises the sequence of SEQ ID NOS: 38and 260-262, 39 and 263-265, 40 and 266-268, and/or 41 and 269-271, andHVR L1, L2, and L3 sequences are SEQ ID NOS:51, 52 and/or 53,respectively. In an additional embodiment, an antibody of the inventioncomprises a light chain variable domain, wherein the framework sequencecomprises the sequence of SEQ ID NOS: 38 and 260-262, 39 and 263-265, 40and 266-268, and/or 41 and 269-271, and HVR L1, L2, and L3 sequences areSEQ ID NOS:87, 88 and/or 89, respectively. In yet another embodiment, anantibody of the invention comprises a light chain variable domain,wherein the framework sequence comprises the sequence of SEQ ID NOS: 38and 260-262, 39 and 263-265, 40 and 266-268, and/or 41 and 269-271, andHVR L1, L2, and L3 sequences are SEQ ID NOS:111, 112, and/or 113,respectively.

In another aspect, an antibody of the invention comprises a heavy chainvariable domain comprising the sequence of SEQ ID NO: 132 and/or a lightchain variable domain comprising the sequence of SEQ ID NO: 133. Inanother aspect, an antibody of the invention comprises a heavy chainvariable domain comprising the sequence of SEQ ID NO: 134 and/or a lightchain variable domain comprising the sequence of SEQ ID NO: 135. Inanother aspect, an antibody of the invention comprises a heavy chainvariable domain comprising the sequence of SEQ ID NO: 136 and/or a lightchain variable domain comprising the sequence of SEQ ID NO: 137. Inanother aspect, an antibody of the invention comprises a heavy chainvariable domain comprising the sequence of SEQ ID NO: 138 and/or a lightchain variable domain comprising the sequence of SEQ ID NO:139.

In one aspect, the invention provides an anti-FGFR3 antibody that bindsa polypeptide comprising, consisting essentially of or consisting of thefollowing amino acid sequence: LAVPAANTVRFRCPA (SEQ ID NO:179) and/orSDVEFHCKVYSDAQP (SEQ ID NO:180).

In some embodiments, the antibody binds a polypeptide comprising,consisting essentially of or consisting of amino acid numbers 164-178and/or 269-283 of the mature human FGFR3 amino acid sequence.

In one embodiment, an anti-FGFR3 antibody of the invention specificallybinds an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%,95%, 98% sequence identity or similarity with the sequenceLAVPAANTVRFRCPA (SEQ ID NO: 179) and/or SDVEFHCKVYSDAQP (SEQ ID NO:180).

In one aspect, the anti-FGFR3 antibody of the present invention binds toat least one, two, three, four, or any number up to all of residues 154,155, 158, 159, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, 175, 177, 202, 205, 207, 210, 212, 214, 216, 217, 241,246, 247, 248, 278, 279, 280, 281, 282, 283, 314, 315, 316, 317 and/or318 of FGFR3 IIIb polypeptide, or equivalent residues of FGFR3 IIIcpolypeptide. One of ordinary skill in the art understands how to alignFGFR3 sequences in order identify corresponding residues betweenrespective FGFR3 sequences. Combinations of two or more residues caninclude any of residues 154, 155, 158, 159, 161, 162, 163, 164, 165,166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 177, 202, 205, 207,210, 212, 214, 216, 217, 241, 246, 247, 248, 278, 279, 280, 281, 282,283, 314, 315, 316, 317 and/or 318 of FGFR3 IIIb polypeptide, orequivalent residues of FGFR3 IIIc polypeptide. In some embodiments, theanti-FGFR3 antibody binds to at least one, two, three, four, or anynumber up to all of residues 158, 159, 169, 170, 171, 173, 175, 205,207, and/or 315 of FGFR3 IIIb polypeptide, or equivalent residues ofFGFR3 IIIc polypeptide. In some embodiments, the anti-FGFR3 antibodybinds to at least one, two three, four, or any number up to all ofresidues 158, 170, 171, 173, 175, and/or 315 of FGFR3 IIIb polypeptide,or equivalent residues of FGFR3 IIIc polypeptide.

In one aspect, the invention provides an anti-FGFR3 antibody thatcompetes with any of the above-mentioned antibodies for binding toFGFR3. In one aspect, the invention provides an anti-FGFR3 antibody thatbinds to the same or a similar epitope on FGFR3 as any of theabove-mentioned antibodies.

As is known in the art, and as described in greater detail hereinbelow,the amino acid position/boundary delineating a hypervariable region ofan antibody can vary, depending on the context and the variousdefinitions known in the art (as described below). Some positions withina variable domain may be viewed as hybrid hypervariable positions inthat these positions can be deemed to be within a hypervariable regionunder one set of criteria while being deemed to be outside ahypervariable region under a different set of criteria. One or more ofthese positions can also be found in extended hypervariable regions (asfurther defined below).

In some embodiments, the antibody is a monoclonal antibody. In otherembodiments, the antibody is a polyclonal antibody. In some embodiments,the antibody is selected from the group consisting of a chimericantibody, an affinity matured antibody, a humanized antibody, and ahuman antibody. In certain embodiments, the antibody is an antibodyfragment. In some embodiments, the antibody is a Fab, Fab′, Fab′-SH,F(ab′)₂, or scFv.

In some embodiment, the FGFR3 antibody is a one-armed antibody (i.e.,the heavy chain variable domain and the light chain variable domain forma single antigen binding arm) comprising an Fc region, wherein the Fcregion comprises a first and a second Fc polypeptide, wherein the firstand second Fc polypeptides are present in a complex and form a Fc regionthat increases stability of said antibody fragment compared to a Fabmolecule comprising said antigen binding arm. See, e.g., WO2006/015371.

In one embodiment, the antibody is a chimeric antibody, for example, anantibody comprising antigen binding sequences from a non-human donorgrafted to a heterologous non-human, human, or humanized sequence (e.g.,framework and/or constant domain sequences). In one embodiment, thenon-human donor is a mouse. In a further embodiment, an antigen bindingsequence is synthetic, e.g., obtained by mutagenesis (e.g., phagedisplay screening, etc.). In a particular embodiment, a chimericantibody of the invention has murine V regions and a human C region. Inone embodiment, the murine light chain V region is fused to a humankappa light chain. In another embodiment, the murine heavy chain Vregion is fused to a human IgG1 C region.

Humanized antibodies of the invention include those that have amino acidsubstitutions in the framework region (FR) and affinity maturationvariants with changes in the grafted CDRs. The substituted amino acidsin the CDR or FR are not limited to those present in the donor orrecipient antibody. In other embodiments, the antibodies of theinvention further comprise changes in amino acid residues in the Fcregion that lead to improved effector function including enhanced CDCand/or ADCC function and B-cell killing. Other antibodies of theinvention include those having specific changes that improve stability.In other embodiments, the antibodies of the invention comprise changesin amino acid residues in the Fc region that lead to decreased effectorfunction, e.g., decreased CDC and/or ADCC function and/or decreasedB-cell killing. In some embodiments, the antibodies of the invention arecharacterized by decreased binding (such as absence of binding) to humancomplement factor C1q and/or human Fc receptor on natural killer (NK)cells. In some embodiments, the antibodies of the invention arecharacterized by decreased binding (such as the absence of binding) tohuman FcγRI, FcγRIIA, and/or FcγRIIIA. In some embodiments, theantibodies of the invention are of the IgG class (e.g., IgG1 or IgG4)and comprise at least one mutation in E233, L234, G236, D265, D270,N297, E318, K320, K322, A327, A330, P331, and/or P329 (numberingaccording to the EU index). In some embodiments, the antibodies comprisethe mutations L234A/L235A or D265A/N297A.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. For example, antibodies with a maturecarbohydrate structure that lacks fucose attached to an Fc region of theantibody are described in US Pat Appl No US 2003/0157108 (Presta, L.).See also US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Antibodies with abisecting N-acetylglucosamine (GlcNAc) in the carbohydrate attached toan Fc region of the antibody are referenced in WO 2003/011878,Jean-Mairet et al. and U.S. Pat. No. 6,602,684, Umana et al. Antibodieswith at least one galactose residue in the oligosaccharide attached toan Fc region of the antibody are reported in WO 1997/30087, Patel et al.See, also, WO 1998/58964 (Raju, S.) and WO 1999/22764 (Raju, S.)concerning antibodies with altered carbohydrate attached to the Fcregion thereof. See also US 2005/0123546 (Umana et al.) onantigen-binding molecules with modified glycosylation. In one aspect,the invention provides FGFR3 binding polypeptides comprising any of theantigen binding sequences provided herein, wherein the FGFR3 bindingpolypeptides specifically bind to a FGFR3, e.g., a human and/or cynoand/or mouse FGFR3.

The antibodies of the invention bind (such as specifically bind) FGFR3(e.g. FGFR3-IIIb and/or FGFR3-IIIc), and in some embodiments, maymodulate (e.g. inhibit) one or more aspects of FGFR3 signaling (such asFGFR3 phosphorylation) and/or disruption of any biologically relevantFGFR3 and/or FGFR3 ligand biological pathway, and/or treatment and/orprevention of a tumor, cell proliferative disorder or a cancer; and/ortreatment or prevention of a disorder associated with FGFR3 expressionand/or activity (such as increased FGFR3 expression and/or activity). Insome embodiments, the FGFR3 antibody specifically binds to a polypeptideconsisting of or consisting essentially of a FGFR3 (e.g., a human ormouse FGFR3). In some embodiments, the antibody specifically binds FGFR3with a Kd of 1×10^(0.7) M or stronger.

In some embodiments, the anti-FGFR3 antibody of the invention is not ananti-FGFR3 antibody described in U.S. Patent Publication no.2005/0147612 (e.g., antibody MSPRO2, MSPRO12, MSPRO59, MSPRO11, MSPRO21,MSPRO24, MSPRO26, MSPRO28, MSPRO29, MSPRO43, MSPRO55), antibodydescribed in Rauchenberger et al, J Biol Chem 278 (40):38194-38205(2003); an antibody described in PCT Publication No. WO2006/048877(e.g., antibody PRO-001), an antibody described inMartinez-Torrecuadrada et al, Mol Cancer Ther (2008) 7(4): 862-873(e.g., scFvαFGFR3 3C), an antibody described in Direnzo, R et al (2007)Proceedings of AACR Annual Meeting, Abstract No. 2080 (e.g., D11), or anantibody described in WO 2010/002862 (e.g., antibodies 15D8, 27H2, 4E7,2G4, 20B4).

In one aspect, the invention provides compositions comprising one ormore antibodies of the invention and a carrier. In one embodiment, thecarrier is pharmaceutically acceptable.

In another aspect, the invention provides nucleic acids encoding a FGFR3antibody of the invention.

In yet another aspect, the invention provides vectors comprising anucleic acid of the invention.

In a further aspect, the invention provides compositions comprising oneor more nucleic acids of the invention and a carrier. In one embodiment,the carrier is pharmaceutically acceptable.

In one aspect, the invention provides host cells comprising a nucleicacid or a vector of the invention. A vector can be of any type, forexample, a recombinant vector such as an expression vector. Any of avariety of host cells can be used. In one embodiment, a host cell is aprokaryotic cell, for example, E. coli. In another embodiment, a hostcell is a eukaryotic cell, for example a mammalian cell such as ChineseHamster Ovary (CHO) cell.

In a further aspect, the invention provides methods of making anantibody of the invention. For example, the invention provides methodsof making an anti-FGFR3 antibody (which, as defined herein includes fulllength antibody and fragments thereof), said method comprisingexpressing in a suitable host cell a recombinant vector of the inventionencoding the antibody, and recovering the antibody. In some embodiments,the method comprises culturing a host cell comprising nucleic acidencoding the antibody so that the nucleic acid is expressed. In someembodiments, the method further comprises recovering the antibody fromthe host cell culture. In some embodiments, the antibody is recoveredfrom the host cell culture medium. In some embodiments, the methodfurther comprises combining the recovered antibody with apharmaceutically acceptable carrier, excipient, or carrier to prepare apharmaceutical formulation comprising the humanized antibody.

In one aspect, the invention provides an article of manufacturecomprising a container; and a composition contained within thecontainer, wherein the composition comprises one or more FGFR3antibodies of the invention. In one embodiment, the compositioncomprises a nucleic acid of the invention. In another embodiment, acomposition comprising an antibody further comprises a carrier, which insome embodiments is pharmaceutically acceptable. In one embodiment, anarticle of manufacture of the invention further comprises instructionsfor administering the composition (e.g., the antibody) to an individual(such as instructions for any of the methods described herein).

In another aspect, the invention provides a kit comprising a firstcontainer comprising a composition comprising one or more anti-FGFR3antibodies of the invention; and a second container comprising a buffer.In one embodiment, the buffer is pharmaceutically acceptable. In oneembodiment, a composition comprising an antibody further comprises acarrier, which in some embodiments is pharmaceutically acceptable. Inanother embodiment, a kit further comprises instructions foradministering the composition (e.g., the antibody) to an individual.

In a further aspect, the invention provides an anti-FGFR3 antibody ofthe invention for use as a medicament.

In a further aspect, the invention provides an anti-FGFR3 antibody ofthe invention for use in treating or preventing a disorder, such as apathological condition associated with FGFR3 activation and/orexpression (in some embodiments, over-expression). In some embodiments,the disorder is a cancer, a tumor, and/or a cell proliferative disorder.In some embodiments, the cancer, a tumor, and/or a cell proliferativedisorder is multiple myeloma or bladder cancer (e.g., transitional cellcarcinoma), breast cancer or liver cancer.

In a further aspect, the invention provides an anti-FGFR3 antibody ofthe invention for use in treating or preventing a disorder such as askeletal disorder. In some embodiments, the disorder is achondroplasia,hypochondroplasia, dwarfism, thantophoric dysplasia (TD; clinical formsTD1 and TDII), or craniosynostosis syndrome.

In a further aspect, the invention provides an anti-FGFR3 antibody ofthe invention for use in reducing cell proliferation.

In a further aspect, the invention provides an anti-FGFR3 antibody ofthe invention for use in killing a cell. In some embodiments, the cellis a multiple myeloma cell. In some embodiments, the cell is killed byADCC. In some embodiments, the antibody is a naked antibody. In someembodiments, the cell over-expresses FGFR3.

In a further aspect, the invention provides an anti-FGFR3 antibody ofthe invention for use in depleting cells, such as multiple myelomacells. In some embodiments, the cell is killed by ADCC. In someembodiments, the antibody is a naked antibody. In some embodiments, thecell over-expresses FGFR3.

In a further aspect, the invention provides use of an anti-FGFR3antibody of the invention in the preparation of a medicament for thetherapeutic and/or prophylactic treatment of a disorder, such as apathological condition associated with FGFR3 activation and/orexpression (in some embodiments, over-expression). In some embodiments,the disorder is a cancer, a tumor, and/or a cell proliferative disorder.In some embodiments, the cancer, a tumor, and/or a cell proliferativedisorder is multiple myeloma or bladder cancer (e.g., transitional cellcarcinoma), breast cancer or liver cancer. In some embodiments, thedisorder is a skeletal disorder, e.g., achondroplasia,hypochondroplasia, dwarfism, thantophoric dysplasia (TD; clinical formsTD1 and TDII), or craniosynostosis syndrome.

In one aspect, the invention provides use of a nucleic acid of theinvention in the preparation of a medicament for the therapeutic and/orprophylactic treatment of a disorder, such as a pathological conditionassociated with FGFR3 activation and/or expression (in some embodiments,over-expression). In some embodiments, the disorder is a cancer, atumor, and/or a cell proliferative disorder. In some embodiments, thecancer, a tumor, and/or a cell proliferative disorder is multiplemyeloma or bladder cancer (e.g., transitional cell carcinoma), breastcancer or liver cancer. In some embodiments, the disorder is a skeletaldisorder, e.g., achondroplasia, hypochondroplasia, dwarfism,thantophoric dysplasia (TD; clinical forms TD1 and TDII), orcraniosynostosis syndrome.

In another aspect, the invention provides use of an expression vector ofthe invention in the preparation of a medicament for the therapeuticand/or prophylactic treatment of a disorder, such as a pathologicalcondition associated with FGFR3 activation and/or expression (in someembodiments, over-expression). In some embodiments, the disorder is acancer, a tumor, and/or a cell proliferative disorder. In someembodiments, the cancer, a tumor, and/or a cell proliferative disorderis multiple myeloma or bladder cancer (e.g., transitional cellcarcinoma), breast cancer or liver cancer. In some embodiments, thedisorder is a skeletal disorder, e.g., achondroplasia,hypochondroplasia, dwarfism, thantophoric dysplasia (TD; clinical formsTD1 and TDII), or craniosynostosis syndrome.

In yet another aspect, the invention provides use of a host cell of theinvention in the preparation of a medicament for the therapeutic and/orprophylactic treatment of a disorder, such as a pathological conditionassociated with FGFR3 activation and/or expression (in some embodiments,over-expression). In some embodiments, the disorder is a cancer, atumor, and/or a cell proliferative disorder. In some embodiments, thecancer, a tumor, and/or a cell proliferative disorder is multiplemyeloma or bladder cancer (e.g., transitional cell carcinoma), breastcancer or liver cancer. In some embodiments, the disorder is a skeletaldisorder, e.g., achondroplasia, hypochondroplasia, dwarfism,thantophoric dysplasia (TD; clinical forms TD1 and TDII), orcraniosynostosis syndrome.

In a further aspect, the invention provides use of an article ofmanufacture of the invention in the preparation of a medicament for thetherapeutic and/or prophylactic treatment of a disorder, such as apathological condition associated with FGFR3 activation and/orexpression (in some embodiments, over-expression). In some embodiments,the disorder is a cancer, a tumor, and/or a cell proliferative disorder.In some embodiments, the cancer, a tumor, and/or a cell proliferativedisorder is multiple myeloma or bladder cancer (e.g., transitional cellcarcinoma), breast cancer or liver cancer. In some embodiments, thedisorder is a skeletal disorder, e.g., achondroplasia,hypochondroplasia, dwarfism, thantophoric dysplasia (TD; clinical formsTD1 and TDII), or craniosynostosis syndrome.

In one aspect, the invention also provides use of a kit of the inventionin the preparation of a medicament for the therapeutic and/orprophylactic treatment of a disorder, such as a pathological conditionassociated with FGFR3 activation and/or expression (in some embodiments,over-expression). In some embodiments, the disorder is a cancer, atumor, and/or a cell proliferative disorder. In some embodiments, thecancer, a tumor, and/or a cell proliferative disorder is multiplemyeloma or bladder cancer (e.g., transitional cell carcinoma), breastcancer or liver cancer. In some embodiments, the disorder is a skeletaldisorder, e.g., achondroplasia, hypochondroplasia, dwarfism,thantophoric dysplasia (TD; clinical forms TD1 and TDII), orcraniosynostosis syndrome.

In a further aspect, the invention provides use of an anti-FGFR3antibody of the invention in the preparation of a medicament forinhibition of cell proliferation. In a further aspect, the inventionprovides use of an anti-FGFR3 antibody of the invention in thepreparation of a medicament for cell killing. In some embodiments, thecell is a multiple myeloma cell. In some embodiments, the cell is killedby ADCC. In some embodiments, the antibody is a naked antibody. In someembodiments, the cell over-expresses FGFR3.

In a further aspect, the invention provides use of an anti-FGFR3antibody of the invention in the preparation of a medicament fordepleting cells, such as multiple myeloma cells. In some embodiments,the cell is killed by ADCC. In some embodiments, the antibody is a nakedantibody. In some embodiments, the cell over-expresses FGFR3.

The invention provides methods and compositions useful for modulatingdisorders associated with expression and/or signaling of FGFR3, such asincreased expression and/or signaling or undesired expression and/orsignaling.

Methods of the invention can be used to affect any suitable pathologicalstate. Exemplary disorders are described herein, and include a cancerselected from the group consisting of non-small cell lung cancer,ovarian cancer, thyroid cancer, testicular cancer, endometrial cancer,head and neck cancer, brain cancer (e.g., neuroblastoma or meningioma),skin cancer (e.g., melanoma, basal cell carcinoma, or squamous cellcarcinoma), bladder cancer (e.g., transitional cell carcinoma), breastcarcinoma, gastric cancer, colorectal cancer (CRC), hepatocellularcarcinoma, cervical cancer, lung cancer, pancreatic cancer, prostatecancer, and hematologic malignancies (e.g., T-cell acute lymphoblasticleukemia (T-ALL), B-cell acute lymphoblastic leukemia (B-ALL), acutemyelogenous leukemia (AML), B-cell malignancies, Hodgkin lymphoma, andmultiple myeloma). In some embodiments, the disorder is invasivetransitional cell carcinoma. In some embodiments, the disorder ismultiple myeloma. Additional exemplary disorders include skeletaldisorders, such as achondroplasia, hypochondroplasia, dwarfism,thantophoric dysplasia (TD; clinical forms TD1 and TDII), orcraniosynostosis syndrome.

In certain embodiments, the cancer expresses FGFR3, amplified FGFR3,translocated FGFR3, and/or mutated FGFR3. In certain embodiments, thecancer expresses activated FGFR3. In certain embodiments, the cancerexpresses translocated FGFR3 (e.g., a t(4; 14) translocation). Incertain embodiments, the cancer expresses constitutive FGFR3. In someembodiments, the constitutive FGFR3 comprises a mutation in the tyrosinekinase domain and/or the juxtamembrane domain and/or a ligand-bindingdomain. In certain embodiments, the cancer expresses ligand-independentFGFR3. In some embodiments, the cancer expresses ligand-dependent FGFR3.

In some embodiments, the cancer expresses FGFR3 comprising a mutationcorresponding to FGFR3-IIIb^(S248C). In some embodiments, the cancerexpressed FGFR3-IIIb^(S248C) and/or FGFR3-IIIc^(S248C).

In some embodiments, the cancer expresses FGFR3 comprising a mutationcorresponding to FGFR3-IIIb^(K652E). In some embodiments, the cancerexpressed FGFR3-IIIb^(K652E) and/or FGFR3-IIIc^(K650E),

FGFR3 comprising a mutation corresponding to FGFR3-IIIb^(S249C). In someembodiments, the cancer expresses FGFR3-IIIb^(S249C) and/orFGFR3-IIIc^(S249C).

In one aspect, the cancer expresses FGFR3 comprising a mutationcorresponding to FGFR3-IIIb^(G372C). In some embodiments, the cancerexpresses FGFR3-IIIb^(G372C) and/or FGFR3-IIIc^(G370C).

In one aspect, the cancer expresses FGFR3 comprising a mutationcorresponding to FGFR3-IIIb^(Y375C). In some embodiments, the cancerexpresses FGFR3-IIIb^(Y375C) and/or FGFR3-IIIc^(Y373C).

In some embodiments, the cancer expresses (a) FGFR3-IIIb^(K652E) and (b)one or more of FGFR3-IIIb^(R248C), FGFR3-IIIb^(Y375C),FGFR3-IIIb^(S249C), and FGFR3IIIb^(G372C).

In some embodiments, the cancer expresses (a) FGFR3-IIIb^(R248C) and (b)one or more of FGFR3-IIIb^(K652E), FGFR3-IIIb^(Y375C),FGFR3-IIIb^(S249C), and FGFR3-IIIb^(G372C).

In some embodiments, the cancer expresses (a) FGFR3-IIIb^(G372C) and (b)one or more of FGFR3-IIIb^(K652E), FGFR3-IIIb^(Y375C),FGFR3-IIIb^(S249C), and FGFR3-IIIb^(R248C).

In some embodiments, the cancer expresses FGFR3-IIIb^(R248C),FGFR3-IIIb^(K652E), FGFR3-IIIb^(Y375C), FGFR3-IIIb^(S249C), andFGFR3-IIIb^(G372C).

In certain embodiments, the cancer expresses increased levels ofphospho-FGFR3, phospho-FRS2 and/or phospho-MAPK relative to a controlsample (e.g., a sample of normal tissue) or level.

In some embodiments, the cancer expresses (e.g., on the cell surface)about 10,000, FGFR3 molecules per cell or more (such as 11,000, 12,000,13,000, 14,000, 15,000, 16,000, 17,000, 18,000 or more FGFR3 receptors).In some embodiments, the cancer expresses about 13000 FGFR3 molecules.In other embodiments, the cancer expresses about 5000, 6000, 7000, 8000,or more FGFR3 molecules. In some embodiments, the cancer expresses lessthan about 4000, 3000, 2000, 1000, or fewer FGFR3 molecules. In someembodiments, the cancer expresses less than about 1000 FGFR3 molecules.

In one embodiment, a cell that is targeted in a method of the inventionis a cancer cell. For example, a cancer cell can be one selected fromthe group consisting of a breast cancer cell, a colorectal cancer cell,a lung cancer cell (e.g., a non-small cell lung cancer cell), a thyroidcancer cell, a multiple myeloma cell, a testicular cancer cell, apapillary carcinoma cell, a colon cancer cell, a pancreatic cancer cell,an ovarian cancer cell, a cervical cancer cell, a central nervous systemcancer cell, an osteogenic sarcoma cell, a renal carcinoma cell, ahepatocellular carcinoma cell, a bladder cancer cell (e.g., atransitional cell carcinoma cell), a gastric carcinoma cell, a head andneck squamous carcinoma cell, a melanoma cell, a leukemia cell, amultiple myeloma cell (e.g. a multiple myeloma cell comprising a t(4:14)FGFR3 translocation) and a colon adenoma cell. In one embodiment, a cellthat is targeted in a method of the invention is a hyperproliferativeand/or hyperplastic cell. In another embodiment, a cell that is targetedin a method of the invention is a dysplastic cell. In yet anotherembodiment, a cell that is targeted in a method of the invention is ametastatic cell.

In one aspect, the invention provides methods for inhibiting cellproliferation in a subject, the method comprising administering to thesubject an effective amount of an anti-FGFR3 antibody to reduce cellproliferation.

In one aspect, the invention provides methods for killing a cell in asubject, the method comprising administering to the subject an effectiveamount of an anti-FGFR3 antibody to kill a cell. In some embodiments,the cell is a multiple myeloma cell. In some embodiments, the cell iskilled by ADCC. In some embodiments, the antibody is a naked antibody.In some embodiments, the cell over-expresses FGFR3.

In one aspect, the invention provides methods for depleting cells (suchas multiple myeloma cells) in a subject, the method comprisingadministering to the subject an effective amount of an anti-FGFR3antibody to kill a cell. In some embodiments, the cell is killed byADCC. In some embodiments, the antibody is a naked antibody. In someembodiments, the cell over-expresses FGFR3.

In one aspect, the invention provides methods for treating or preventinga skeletal disorder. In some embodiments, the disorder isachondroplasia, hypochondroplasia, dwarfism, thantophoric dysplasia (TD;clinical forms TD1 and TDII), or craniosynostosis syndrome.

Methods of the invention can further comprise additional treatmentsteps. For example, in one embodiment, a method further comprises a stepwherein a targeted cell and/or tissue (e.g., a cancer cell) is exposedto radiation treatment or a chemotherapeutic agent.

In one aspect, the invention provides methods comprising administrationof an effective amount of an anti-FGFR3 antibody in combination with aneffective amount of another therapeutic agent (such as ananti-angiogenesis agent, another antibody, a chemotherapeutic agent, acytotoxic agent, an immunosuppressive agent, a prodrug, a cytokine,cytotoxic radiotherapy, a corticosteroid, an anti-emetic, a cancervaccine, an analgesic, or a growth inhibitory agent). For example,anti-FGFR3 antibodies are used in combinations with an anti-cancer agentor an anti-angiogenic agent to treat various neoplastic ornon-neoplastic conditions.

In particular examples, the anti-FGFR3 antibodies are used incombination with velcade, revlimid, tamoxifen, letrozole, exemestane,anastrozole, irinotecan, cetuximab, fulvestrant, vinorelbine,bevacizumab, vincristine, cisplatin, gemcitabine, methotrexate,vinblastine, carboplatin, paclitaxel, docetaxel, pemetrexed,5-fluorouracil, doxorubicin, bortezomib, lenalidomide, dexamethasone,melphalin, prednisone, vincristine, and/or thalidomide.

Depending on the specific cancer indication to be treated, thecombination therapy of the invention can be combined with additionaltherapeutic agents, such as chemotherapeutic agents, or additionaltherapies such as radiotherapy or surgery. Many known chemotherapeuticagents can be used in the combination therapy of the invention.Preferably those chemotherapeutic agents that are standard for thetreatment of the specific indications will be used. Dosage or frequencyof each therapeutic agent to be used in the combination is preferablythe same as, or less than, the dosage or frequency of the correspondingagent when used without the other agent(s).

In another aspect, the invention provides any of the anti-FGFR3antibodies described herein, wherein the anti-FGFR3 antibody comprises adetectable label.

In another aspect, the invention provides a complex of any of theanti-FGFR3 antibodies described herein and FGFR3. In some embodiments,the complex is in vivo or in vitro. In some embodiments, the complexcomprises a cancer cell. In some embodiments, the anti-FGFR3 antibody isdetectably labeled.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B and 1C: Heavy chain and light chain HVR loop sequences ofanti-FGFR3 antibodies. The figures show the heavy chain HVR sequences,H1, H2, and H3, and light chain HVR sequences, L1, L2, and L3. Sequencenumbering is as follows:

Clone 184.6 (HVR-H1 is SEQ ID NO:1; HVR-H2 is SEQ ID NO:2; HVR-H3 is SEQID NO:3; HVR-L1 is SEQ ID NO:4; HVR-L2 is SEQ ID NO:5; HVR-L3 is SEQ IDNO:6);

Clone 184.6.1 (HVR-H1 is SEQ ID NO:7; HVR-H2 is SEQ ID NO:8; HVR-H3 isSEQ ID NO:9; HVR-L1 is SEQ ID NO:10; HVR-L2 is SEQ ID NO: 11; HVR-L3 isSEQ ID NO:12)

Clone 184.6.58 (HVR-H1 is SEQ ID NO:13; HVR-H2 is SEQ ID NO:14; HVR-H3is SEQ ID NO:15; HVR-L1 is SEQ ID NO:16; HVR-L2 is SEQ ID NO:17; HVR-L3is SEQ ID NO:18)

Clone 184.6.62 (HVR-H1 is SEQ ID NO:48; HVR-H2 is SEQ ID NO:49; HVR-H3is SEQ ID NO:50; HVR-L1 is SEQ ID NO:51; HVR-L2 is SEQ ID NO:52; HVR-L3is SEQ ID NO:53)

Clone 184.6.21 (HVR-H1 is SEQ ID NO:54; HVR-H2 is SEQ ID NO:55; HVR-H3is SEQ ID NO:56; HVR-L1 is SEQ ID NO:57; HVR-L2 is SEQ ID NO:58; HVR-L3is SEQ ID NO:59)

Clone 184.6.49 (HVR-H1 is SEQ ID NO:60; HVR-H2 is SEQ ID NO:61; HVR-H3is SEQ ID NO:62; HVR-L1 is SEQ ID NO:63; HVR-L2 is SEQ ID NO:64; HVR-L3is SEQ ID NO:65)

Clone 184.6.51 (HVR-H1 is SEQ ID NO:66; HVR-H2 is SEQ ID NO:67; HVR-H3is SEQ ID NO:68; HVR-L1 is SEQ ID NO:69; HVR-L2 is SEQ ID NO:70; HVR-L3is SEQ ID NO:71)

Clone 184.6.52 (HVR-H1 is SEQ ID NO:72; HVR-H2 is SEQ ID NO:73; HVR-H3is SEQ ID NO:74; HVR-L1 is SEQ ID NO:75; HVR-L2 is SEQ ID NO:76; HVR-L3is SEQ ID NO:77)

Clone 184.6.92 (HVR-H1 is SEQ ID NO:78; HVR-H2 is SEQ ID NO:79; HVR-H3is SEQ ID NO:80; HVR-L1 is SEQ ID NO:81; HVR-L2 is SEQ ID NO:82; HVR-L3is SEQ ID NO:83)

Clone 184.6.1.N54S (HVR-H1 is SEQ ID NO:84; HVR-H2 is SEQ ID NO:85;HVR-H3 is SEQ ID NO:86; HVR-L1 is SEQ ID NO:87; HVR-L2 is SEQ ID NO:88;HVR-L3 is SEQ ID NO:89)

Clone 184.6.1.N54G (HVR-H1 is SEQ ID NO:90; HVR-H2 is SEQ ID NO:91;HVR-H3 is SEQ ID NO:92; HVR-L1 is SEQ ID NO:93; HVR-L2 is SEQ ID NO:94;HVR-L3 is SEQ ID NO:95)

Clone 184.6.1.N54A (HVR-H1 is SEQ ID NO:96; HVR-H2 is SEQ ID NO:97;HVR-H3 is SEQ ID NO:98; HVR-L1 is SEQ ID NO:99; HVR-L2 is SEQ ID NO:100;HVR-L3 is SEQ ID NO:101)

Clone 184.6.1.N54Q (HVR-H1 is SEQ ID NO:102; HVR-H2 is SEQ ID NO:103;HVR-H3 is SEQ ID NO: 104; HVR-L1 is SEQ ID NO: 105; HVR-L2 is SEQ ID NO:106; HVR-L3 is SEQ ID NO: 107)

Clone 184.6.58.N54S (HVR-H1 is SEQ ID NO:108; HVR-H2 is SEQ ID NO:109;HVR-H3 is SEQ ID NO: 110; HVR-L1 is SEQ ID NO: 111; HVR-L2 is SEQ ID NO:112; HVR-L3 is SEQ ID NO: 113)

Clone 184.6.58.N54G (HVR-H1 is SEQ ID NO: 114; HVR-H2 is SEQ ID NO: 115;HVR-H3 is SEQ ID NO: 116; HVR-L1 is SEQ ID NO: 117; HVR-L2 is SEQ ID NO:118; HVR-L3 is SEQ ID NO: 119)

Clone 184.6.58.N54A (HVR-H1 is SEQ ID NO:120; HVR-H2 is SEQ ID NO:121;HVR-H3 is SEQ ID NO: 122; HVR-L1 is SEQ ID NO:123; HVR-L2 is SEQ IDNO:124; HVR-L3 is SEQ ID NO:125)

Clone 184.6.58.N54Q (HVR-H1 is SEQ ID NO:126; HVR-H2 is SEQ ID NO:127;HVR-H3 is SEQ ID NO: 128; HVR-L1 is SEQ ID NO: 129; HVR-L2 is SEQ ID NO:130; HVR-L3 is SEQ ID NO:131).

Clone 184.6.1.NS D30E (HVR-H1 is SEQ ID NO:143; HVR-H2 is SEQ ID NO:144;HVR-H3 is SEQ ID NO:145; HVR-L1 is SEQ ID NO:140; HVR-L2 is SEQ IDNO:141; HVR-L3 is SEQ ID NO: 142).

Amino acid positions are numbered according to the Kabat numberingsystem as described below.

FIGS. 2A and 2B: depict (A) the amino acid sequences of the heavy chainvariable regions and light chain variable regions of anti-FGFR3antibodies 184.6.1.N54S, 184.6.58, and 184.6.62; and (B) thehypervariable regions of anti-FGFR3 antibodies 1G6, 6G1, and 15B2.

FIGS. 3A, 3B, and 4: depict exemplary acceptor human consensus frameworksequences for use in practicing the instant invention with sequenceidentifiers as follows:

Variable heavy (VH) consensus frameworks (FIG. 3A, 3B)

human VH subgroup I consensus framework minus Kabat CDRs (SEQ ID NOS:19and 203-205) human VH subgroup I consensus framework minus extendedhypervariable regions (SEQ ID NOS:20 and 206-208, 21 and 209-211, 22 and212-214) human VH subgroup II consensus framework minus Kabat CDRs (SEQID NOS:23 and 215-217)

human VH subgroup II consensus framework minus extended hypervariableregions (SEQ ID NOS:24 and 218-220, 25 and 221-223, 26 and 224-226)human VH subgroup II consensus framework minus extended

human VH subgroup III consensus framework minus Kabat CDRs (SEQ IDNOS:27 and 227-229)

human VH subgroup III consensus framework minus extended hypervariableregions (SEQ ID NOS:28 and 230-232, 29 and 233-235, 30 and 236-238)

human VH acceptor framework minus Kabat CDRs (SEQ ID NOS:31 and 239-241)

human VH acceptor framework minus extended hypervariable regions (SEQ IDNOS:32 and 242-244, 33 and 2245-247)

human VH acceptor 2 framework minus Kabat CDRs (SEQ ID NOS:34 and248-250)

human VH acceptor 2 framework minus extended hypervariable regions (SEQID NOS:35 and 251-253, 36 and 254-256, 37 and 257-259)

Variable light (VL) consensus frameworks (FIG. 4)

human VL kappa subgroup I consensus framework (SEQ ID NO:38 and 260-262)

human VL kappa subgroup II consensus framework (SEQ ID NO:39 and263-265)

human VL kappa subgroup III consensus framework (SEQ ID NO:40 and266-268)

human VL kappa subgroup IV consensus framework (SEQ ID NO:41 and269-271)

FIG. 5: depicts framework region sequences of huMAb4D5-8 light (SEQ IDNOS:42-45) and heavy chains (SEQ ID NOS:46, 47, 175, 176). Numbers insuperscript/bold indicate amino acid positions according to Kabat.

FIG. 6: depicts modified/variant framework region sequences ofhuMAb4D5-8 light (SEQ ID NOS:42, 43, 177, 45) and heavy chains (SEQ IDNOS:46, 47, 178, and 176). Numbers in superscript/bold indicate aminoacid positions according to Kabat.

FIGS. 7A, 7B, 7C and 7D: FGFR3 knockdown in bladder cancer cell RT112inhibits proliferation and induces G1 cell cycle arrest in vitro, andsuppresses tumor growth in vivo. Three different FGFR3 shRNAs werecloned into a Tet-inducible expression vector. RT112 cells stablyexpressing FGFR3 shRNAs or a control shRNA were established withpuromycin selection. (FIG. 7A) Representative blots showing FGFR3expression in selected clones treated with or without doxycycline (Dox,0, 0.1 and 1 μg/ml, left to right). (FIG. 7B) [³H]-thymidineincorporation by RT112 stable cells. RT112 stable clones were culturedwith or without 1 μg/ml doxycycline for 3 days prior to 16hour-incubation with [³H]-thymidine (1 μCi per well). Counts ofincorporated [³H]-thymidine were normalized to that from cells withoutdoxycycline induction. Error bars represent SEM. (FIG. 7C) DNAfluorescence flow cytometry histograms of RT112 stable cells. RT112clones expressing control shRNA or FGFR3 shRNA4 were cultured with orwithout 1 μg/ml doxycycline for 72 hours, and the nuclei were stainedwith propidium iodide (PI). Similar results were obtained for FGFR3shRNA2 and 6 (FIG. 16). (FIG. 7D) The growth of RT112 cells expressingcontrol shRNA (n=9 per treatment group) or FGFR3 shRNA4 (n=1 pertreatment group) in mice. Mice were given 5% sucrose alone orsupplemented with 1 mg/ml doxycycline, and tumor size was measured twicea week. Error bars represent SEM. Similar results were obtained forFGFR3 shRNA2 and 6 (FIG. 16). Lower panel: Expression of FGFR3 proteinin tumor lysates extracted from control shRNA or FGFR3 shRNA4 stablecell xenograft tissues.

FIGS. 8A, 8B, 8C, 8D, and 8E: R3Mab blocks FGF/FGFR3 interaction. (FIG.8A) Selective binding of human FGFR3 by R3Mab. Human FGFR1-4 Fc chimericproteins were immobilized and incubated with increasing amount of R3Mab.Specific binding was detected using an anti-human Fab antibody. (FIGS.8B-8C) Blocking of FGF1 binding to human FGFR3-IIIb (FIG. 8B) or IIIc(FIG. 8C) by R3Mab. Specific binding was detected by using abiotinylated FGF 1-specific polyclonal antibody. (FIGS. 8D-8E) Blockingof FGF9 binding to human FGFR3-IIIb (FIG. 8D) or IIIc (FIG. 8E) byR3Mab. Specific binding was detected by using a biotinylatedFGF9-specific polyclonal antibody. Error bars represent standard errorof the mean (SEM) and are sometimes smaller than symbols.

FIGS. 9A, 9B, 9C, 9D, 9E, 9F, 9G, and 9H: R3Mab inhibits Ba/F3 cellproliferation driven by wild type and mutated FGFR3. (FIG. 9A)Inhibitory effect of R3Mab on the viability of Ba/F3 cells expressingwild type human FGFR3-IIIb. Cells were cultured in medium without FGF1(no FGF1), or in the presence of 10 ng/ml FGF1 plus 10 μg/ml heparinalone (FGF 1), or in combination with a control antibody (Control) orR3Mab. Cell viability was assessed with CellTiter-Glo (Promega) after 72hr incubation with antibodies. (FIG. 9B) Inhibition of FGFR3 and MAPKphosphorylation by R3Mab in Ba/F3-FGFR3-IIIb^(WT) stable cells. Cellswere treated with 15 ng/ml FGF 1 and 10 μg/ml heparin (+) or heparinalone (−) for 10 minutes, following pre-incubation with a Control Ab(Ctrl), decreasing amount of R3Mab (1, 0, 2, 0.04 μg/ml respectively) inPBS, or PBS alone (Mock) for 3 hours. Lysates were immunoblotted toassess phosphorylation of FGFR3 and p44/42 MAPK with antibodies topFGFR^(Y653/654) and pMAPK^(Thr202/Tyr204) respectively. (FIG. 9C)Schematic representation of FGFR3 mutation hot spots and frequency inbladder cancer (sequence numbering depicted is based on the FGFR3 IIIbisoform amino acid sequence) based on published data (32). TM,transmembrane domain; TK1 and TK2, tyrosine kinase domain 1 and 2.(FIGS. 9D-9H) Inhibitory effect of R3Mab on the viability of Ba/F3 cellsexpressing cancer-associated FGFR3 mutants. G372C is derived from IIIcisoform, and the rest are derived from IIIb isoform. Sequence numberingfor all mutants is based on the FGFR3 IIIb isoform amino acid sequence(including the G372C mutant, which would be numbered G370C based on theFGFR3 IIIc isoform amino acid sequence). Cell viability was assessedafter 72 hour incubation with antibodies as described in (FIG. 9A).Error bars represent SEM.

FIGS. 10A, 10B, 10C, 10D, 10E, and 10F: Epitope mapping for R3Mab andcrystal structure of the complex between R3Mab Fab fragment and IgD2-D3of human FGFR3-IIIb. (FIG. 10A) Epitope determined by the binding of 13peptides spanning IgD2-D3 of human FGFR3 to R3Mab. Each biotinylatedpeptide was captured onto streptavidin-coated microtiter well andincubated with R3Mab. Specifically bound R3Mab was detected using a goatanti-human IgG antibody. (FIG. 10B) Sequence alignment of human FGFR3peptides 3 (LAVPAANTVRFRCPA (SEQ ID NO:179) and 11 (SDVEFHCKVYSDAQP (SEQID NO:180) with extracellular segments of human FGFR1 (peptide 3:HAVPAAKTVKFKCPS (SEQ ID NO:181); peptide 11: SNVEFMCKVYSDPQP (SEQ IDNO:182)). FGFR1 residues engaged in the primary FGF2-FGFR1 interaction,heparin binding, and receptor-receptor association are shown in bold,italics, and underlined font, respectively. Functional assignment ofFGFR1 residues is based on Plotnikov et al (34). (FIG. 10C) Structure ofR3Mab Fab (shown in ribbon-helix, light chain grey, heavy chain black)in complex with human FGFR3 IgD2-D3 (shown in molecular surface, white).Receptor residues involved in ligand binding and dimerization arecolored in grey/crosshatched and dark grey respectively based onPlotnikov et al (34). (FIG. 10D) The close-up of the crystal structureshows that CDR-H3 and -H2 from the Fab constitute the major interactionsites with IgD2 and IgD3 of FGFR3. (FIG. 10E) Superposition ofFGFR3-IIIc-FGF1 complex (PDB code 1RY7) with FGFR3-IIIb-Fab complex.FGFR3-IIIc and FGF1 are colored in grey and dark grey respectively.FGFR3-IIIb is shown in white and the Fab is shown in light grey forlight chain, dark grey for heavy chain. IgD2 was used as the anchor forsuperposition. Note the well-superposed IgD2 from both structures andthe new conformation adopted by IgD3 of FGFR3-IIIb when bound by R3Mab.(FIG. 10F) Another representation of the superposition ofFGFR3-IIIc-FGF1 complex (PDB code 1RY7) with FGFR3-IIIb-Fab complex.FGFR3-IIIc and FGF1 are shown as molecular surfaces that are grey/meshtexture and dark grey/dotted texture, respectively. FGFR3-IIIb is shownin white and the Fab is shown in grey for light chain, black for heavychain. IgD2 was used as the anchor for superposition. Note thewell-superposed IgD2 from both structures and the new conformationadopted by IgD3 of FGFR3-IIIb when bound by R3Mab.

FIGS. 11A, 11B, 11C, 11D and 11E: R3Mab inhibits proliferation, clonalgrowth and FGFR3 signaling in bladder cancer cells expressing wild typeor mutated FGFR3^(S249C) (FIG. 11A) Inhibition of [³H]-thymidineincorporation by R3Mab in bladder cancer cell line RT112. Error barsrepresent SEM. (FIG. 11B) Blocking of FGF1-activated FGFR3 signaling byR3Mab (15 μg/ml) in bladder cancer cell line RT112 as compared totreatment medium alone (Mock) or a control antibody (Ctrl). Cell lysateswere immunoprecipitated with anti-FGFR3 antibody and assessed for FGFR3phosphorylation with an anti-phospho-tyrosine antibody (4G10). Lysateswere immunoblotted to detect phosphorylation of AKT (pAKT^(S473)) andp44/42 MAPK (pMAPK^(Thr202/Tyr204)). (FIG. 11C) Inhibition of clonalgrowth by R3Mab (10 μg/ml) in bladder cancer cell line UMUC-14(harboring FGFR3^(S249C)) as compared to treatment medium alone (Mock)or a control antibody (Ctrl). (FIG. 11D) Quantitation of the study in(C) reporting the number of colonies larger than 120 μm in diameter perwell from a replicate of 12 wells. Error bars represent SEM. P<3.4×10⁻⁹versus Mock or Ctrl. (FIG. 11E) Inhibition of FGFR3 phosphorylation inUMUC-14 cells by R3Mab (15 μg/ml). FGFR3 phosphorylation was analyzed asin (B). Note constitutive phosphorylation of FGFR3 in this cell line.

FIGS. 12A, 12B and 12C: R3Mab decreases steady-state level ofdisulfide-linked FGFR3^(S249C) dimer by driving the dimer-monomerequilibrium toward monomeric state. (FIG. 12A) Effect of R3Mab onFGFR3^(S249C) dimer in UMUC-14 cells. Cells were incubated with R3Mab(15 μg/ml) or a control antibody (Ctrl) for 3 hours, and whole celllysates were analyzed by immunoblot under non-reducing and reducingconditions. (FIG. 12B) Effect of free-sulfhydryl blocker DTNB onFGFR3^(S249C) dimer-monomer equilibrium in UMUC-14 cells. UMUC-14 cellswere treated with increasing concentration of DTNB for 3 hours, and celllysates were analyzed as in (FIG. 12A). (FIG. 12C) Effect of R3Mab onpurified recombinant FGFR3^(S249C) dimer in vitro. FGFR3^(S249C) dimercomposed of IgD2-D3 was purified through size-exclusion column, andincubated with PBS (Mock), a control antibody (Ctrl), or R3Mab at 37° C.Samples were collected at indicated time for immunoblot analysis undernon-reducing conditions. FGFR3 dimer-monomer was detected usinganti-FGFR3 hybridoma antibody 6G1 (FIGS. 12A-C).

FIGS. 13A, 13B, 13C, 13D and 13E: R3Mab inhibits xenograft growth ofbladder cancer cells and allograft growth of Ba/F3-FGFR3^(S249C). (FIG.13A) Effect of R3Mab on the growth of pre-established RT112 bladdercancer xenografts compared with vehicle control. n=10 per group. (FIG.13B) Inhibition of FGFR3 signaling in RT112 tumor tissues by R3Mab. In aseparate experiment, RT112 xenograft tumors that were treated with 15mg/kg of a control antibody (Ctrl) or R3Mab for 48 hours or 72 hourswere collected (n=3 per group), homogenized and analyzed for FRS2c andMAPK activation by immunoblot. (FIG. 13C) Effect of R3Mab on the growthof pre-established Ba/F3-FGFR3^(S249C) allografts. n=10 per group. (FIG.13D) Effect of R3Mab on the growth of pre-established UMUC-14 bladdercancer xenografts, n=10 per group. (FIG. 13E) Effect of R3Mab onFGFR3^(S249C) dimer and signaling in UMUC-14 tumor tissues. UMUC-14xenograft tumors that were treated with 30 mg/kg of a control antibody(Ctrl) or R3Mab for 24 hours or 72 hours were collected (n=3 per group),homogenized, and analyzed for FGFR3^(S249C) dimer-monomer as well asMAPK activation by immunoblot. FGFR3 dimer-monomer was detected using ananti-FGFR3 rabbit polyclonal antibody sc9007 to avoid interference frommouse IgG in tumor lysates. Error bars represent SEM.

FIGS. 14A, 14B, 14C, 14D, 14E, 14F, 14G, and 14H: ADCC contributes tothe anti-tumor efficacy of R3Mab in t(4;14) positive multiple myelomamodels. (FIGS. 14A-14B) Effect of R3Mab on the growth of pre-establishedOPM2 (FIG. 14A) and KMS11 (FIG. 14B) myeloma xenografts. n=10 per group.(FIGS. 14C-14F) Cytolysis of myeloma cell lines OPM2 (FIG. 14C) andKMS11 (FIG. 14D), or bladder cancer cell lines RT112 (FIG. 14E) andUMUC-14 (FIG. 14F) induced by R3Mab in cell culture. Myeloma or bladdercancer cells were incubated with freshly isolated human PBMC in thepresence of R3Mab or a control antibody. Cytotoxicity was determined bymeasuring LDH released in the supernatant. (FIGS. 14G-14H) Effect ofR3Mab or its DANA mutant on the growth of pre-established OPM2 (FIG.14G) and KMS11 (FIG. 14H) myeloma xenografts. n=10 per group. Error barsrepresent SEM and are sometimes smaller than symbols.

FIGS. 15A, 15B, 15C and 15D: Knockdown of FGFR3 with siRNA inhibits cellproliferation of bladder cancer cell lines. Six to seven different FGFR3siRNAs and three nonspecific control siRNAs were designed andsynthesized in Genentech. Bladder cancer cell lines RT112 (FIG. 15A),SW780 (FIG. 15B), RT4 (FIG. 15C) and UMUC-14 (FIG. 15D) were plated into96-well plate (3000 cells per well) and allowed to attach overnight, andtransiently transfected with 25 nM siRNA in complex with RNAiMax(Invitrogen). 72 hr post-transfection, [³H]-thymidine (1 μCi per well)was added to the culture (FIGS. 15A, 15C, and 15D) for another 16 hourincubation. Incorporated [³H]-thymidine was quantitated with TopCount.Data were normalized to that from cells transfected with RNAiMax alone(Mock). Error bars represent SEM. Lower panel: Representative blotsshowing FGFR3 expression in siRNA transfected cells. (FIG. 15B) Cellviability was measured with CellTiter-Glo (Promega) 96 hours aftertransfection. Error bars represent SEM.

FIGS. 16A and 16B: FGFR3 knockdown in bladder cancer cell line RT112induces G1 cell cycle arrest in vitro, and suppresses tumor growth invivo. Three different FGFR3 RNAs were designed and cloned into aTet-inducible shRNA expression retroviral vector. RT112 stable clonesexpressing FGFR3 shRNAs or control shRNA were established with puromycinselection. (FIG. 16A) DNA fluorescence flow cytometry histograms ofpropidium iodide (PI)-stained nuclei obtained from RT112 stable cellsexpressing FGFR3 shRNA2 or shRNA6 following treatment with or without 1μg/ml doxycycline for 72 hours. (FIG. 16B) The growth of RT112 stablecells expressing FGFR3 shRNA2-4 (n=1 per treatment group) orFGFR3shRNA6-16 (n=10 per treatment group) in nu/nu mice. Tumor bearingmice received 5% sucrose only (solid circle) or 5% sucrose plus 1 mg/mldoxycycline (solid square), and tumors were measured with calipers twicea week. Error bars represent SEM.

FIG. 17: Effect of anti-FGFR3 hybridoma antibodies 16G, 6G1 and 15B2 onBa/F3 cell proliferation driven by wild type and mutated FGFR3.Anti-FGFR3 hybridoma antibodies were generated by immunizing BALB/c micewith human FGFR3-IIIb/Fc or human FGFR3-IIIc/Fc chimera. Fused hybridomacells were selected using hypoxanthin-aminopterin-thymidine selection inMedium D from the ClonaCell® hybridoma selection kit (StemCellTechnologies, Inc., Vancouver, BC, Canada). Hybridoma antibodies weresequentially screened for their ability to bind to FGFR3-IIIb andFGFR3-IIIc by ELISA and to recognize cell surface FGFR3 by FACS.Selected hybridomas were then cloned by limiting dilution. 16G, 6G1 and15B2 are clones used to assess the effect on the proliferation of Ba/F3cells expressing wild type or mutated FGFR3 similarly as described inFIG. 9A. Error bars represent SEM.

FIGS. 18A and 18B: Comparison of R3Mab epitopes determined by peptidemapping and crystal structure analysis. (FIG. 18A) Epitope revealed bythe structure of the R3Mab Fab fragment in complex with theextracellular IgD2-D3 segment of human FGFR3. FGFR3 residues contactedby Fab heavy chain and light chain are colored in black and grey,respectively. (FIG. 18B) Location of peptides 3 and 11 on FGFR3.

FIGS. 19A, 19B, 19C, 19D and 19E: R3Mab inhibits proliferation and FGFR3signaling in bladder cancer cells containing wild type or mutatedFGFR3^(S249C). (FIG. 19A) Inhibition of cell viability by R3Mab inbladder cancer cell line RT4. Cell viability was assessed withCellTiter-Glo (Promega) after 96 hr incubation with the antibody. Errorbars represent SEM. (FIG. 19B) Blocking of FGF1-activated FGFR3signaling by R3Mab (15 ug/ml) in bladder cancer cell line RT4. (FIG.19C) Inhibition of [³H]-thymidine incorporation by R3Mab in bladdercancer cell line RCC-97-7 (containing FGFR3^(S249C)). Error barsrepresent SEM. (FIG. 19D) Inhibition of FGFR3 phosphorylation inTCC-97-7 cells by R3Mab (15 ug/ml). (FIG. 19E) Decrease of FGFR3^(S249C)dimer in TCC-97-7 cells after 3 hours incubation with R3Mab (15 ug/ml)compared with a control antibody (Ctrl).

FIGS. 20A and 20B: Effect of endocytosis inhibitors on theinternalization of R3Mab and FGFR3^(S249C) dimer in UMUC-14 cells. (FIG.20A) Effect of endocytosis inhibitors on the internalization of R3Mab.UMUC-14 cells, pre-treated with various endocytosis inhibitor or DMSOfor 1 hour at 37° C., were incubated with R3Mab (15 ug/ml) for 3 hoursat 37° C. to allow internalization. A low pH wash was used to removecell surface R3Mab to visualize internalized antibody. Cells were fixedand stained with Alexa 488-labeled anti-human IgG. Image was taken usingconfocal microscopy. (FIG. 20B) Effect of endocytosis inhibitors onFGFR3^(S249C) dimer in UMUC-14 cells treated with R3Mab. UMUC-14 cells,pre-treated with various endocytosis inhibitor or DMSO for 1 hour at 37°C., were incubated with mock (Lane 1), a control antibody (Lane 2), orR3Mab (15 ug/ml, Lane 3) for 3 hours at 37° C. Cell lysates wereanalyzed for FGFR3 protein under non-reducing or reducing conditions byimmunoblot. Note that chlorpromazine (inhibitor of clathrin-mediatedendocytosis) and genistein (pan-inhibitor of endocytosis) blocked R3Mabinternalization, but had no effect on R3Mab-induced decrease ofFGFR3^(S249C) dimer.

FIG. 21: Detection sensitivity of different anti-FGFR3 antibodies towardmonomeric and dimeric FGFR3^(S249C) under non-reducing conditions.UMUC-14 cells were lysed after treatment with R3Mab (Lane 1), a controlIgG1 (Lane 2), or PBS (Lane 3) for 3 hours, and cell lysates weresubject to immunoblot analyses under reducing or non-reducingconditions. Note that 6G1 (murine hybridoma antibody generated atGenentech) detected both FGFR3^(S249C) dimer and monomer, whereas sc9007(rabbit polyclonal antibody, Santa Cruz Biotechnology) or sc13121(murine hybridoma antibody, Santa Cruz Biotechnology) preferentiallydetected the dimeric FGFR3^(S249C).

FIGS. 22A, 22B and 22C: Effect of R3Mab on the proliferation of t(4;14)+ multiple myeloma cells. (FIG. 22A) Inhibitory effect of R3Mab on[³H]-thymidine incorporation by UTMC-2 cells. UTMC-2 cells were grown inmedium containing R3Mab or a control antibody in the presence of 25ng/ml FGF9 and 5 ug/ml heparin or heparin alone (No FGF9). After 6 daysincubation, [³H]-thymidine was added for 16 hr incubation. Data werenormalized to that from cells grown in the absence of FGF9 and antibody.(FIGS. 22B-22C) Effect of R3Mab on [³H]-thymidine incorporation by OPM2(FIG. 22B) and KMS11 (FIG. 22C) cells. Cells grown in 1.5% FBS mediumwere treated with R3Mab or a control antibody for 6 days. Data werenormalized to that from untreated cells. Error bars represent SEM.

FIGS. 23A and 23B: Cell surface expression levels of FGFR3 in myelomaand bladder cancer cells. (FIG. 23A) Cell surface FGFR3 expression inmyeloma cells and bladder cancer cells assessed by FACS analysis. Cellswere stained with phycoerythin-conjugated mouse mAb against human FGFR3(FAB766P, R&D Systems) or phycoerythin-conjugated isotype control mouseIgG1 (BD Pharmingen). (FIG. 23B) Scatchard analysis of FGFR3 density inmyeloma cells and bladder cancer cells. R3Mab was radioiodinated, andincubated with cells in suspension with excess unlabeled antibody. Afterincubation at RT for 2 hours, cells were pelleted by centrifugation andwashed twice. Specifically bound ¹²⁵I was determined. Receptor densityand binding affinity (Kd) represent the mean from two bindingexperiments.

FIGS. 24A and 24B: Effect of R3Mab or its DANA mutant on xenograftgrowth of bladder carcinoma cells. (FIG. 24A) Effect of R3Mab and itsDANA mutant (50 mg/kg each) on the growth of pre-established RT112tumors. (FIG. 24B) Effect of R3Mab and its DANA mutant (50 mg/kg each)on the growth of pre-established UMUC-14 tumors. Error bars representSEM.

DETAILED DESCRIPTION OF THE INVENTION

The invention herein provides anti-FGFR3 antibodies that are useful for,e.g., treatment or prevention of disease states associated withexpression and/or activity of FGFR3, such as increased expression and/oractivity or undesired expression and/or activity. In some embodiments,the antibodies of the invention are used to treat a tumor, a cancer,and/or a cell proliferative disorder.

In another aspect, the anti-FGFR3 antibodies of the invention findutility as reagents for detection and/or isolation of FGFR3, such asdetection of FGFR3 in various tissues and cell type. The inventionfurther provides methods of making and using anti-FGFR3 antibodies, andpolynucleotides encoding anti-FGFR3 antibodies.

General Techniques

The techniques and procedures described or referenced herein aregenerally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized methodologies described in Sambrook et al., MolecularCloning: A Laboratory Manual 3rd. edition (2001) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. CURRENT PROTOCOLS INMOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (2003)); the seriesMETHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICALAPPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)),Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMALCELL CULTURE (R. I. Freshney, ed. (1987)).

Definitions

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gelelectrophoresis) under reducing or nonreducing conditions usingCoomassie blue or, preferably, silver stain. Isolated antibody includesthe antibody in situ within recombinant cells since at least onecomponent of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe nucleic acid. An isolated nucleic acid molecule is other than in theform or setting in which it is found in nature. Isolated nucleic acidmolecules therefore are distinguished from the nucleic acid molecule asit exists in natural cells. However, an isolated nucleic acid moleculeincludes a nucleic acid molecule contained in cells that ordinarilyexpress the nucleic acid (for example, an antibody encoding nucleicacid) where, for example, the nucleic acid molecule is in a chromosomallocation different from that of natural cells.

The term “variable domain residue numbering as in Kabat” or “amino acidposition numbering as in Kabat,” and variations thereof, refers to thenumbering system used for heavy chain variable domains or light chainvariable domains of the compilation of antibodies in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991). Using thisnumbering system, the actual linear amino acid sequence may containfewer or additional amino acids corresponding to a shortening of, orinsertion into, a FR or CDR of the variable domain. For example, a heavychain variable domain may include a single amino acid insert (residue52a according to Kabat) after residue 52 of H2 and inserted residues(e.g. residues 82a, 82b, and 82c, etc according to Kabat) after heavychain FR residue 82. The Kabat numbering of residues may be determinedfor a given antibody by alignment at regions of homology of the sequenceof the antibody with a “standard” Kabat numbered sequence.

The phrase “substantially similar,” or “substantially the same,” as usedherein, denotes a sufficiently high degree of similarity between twonumeric values (generally one associated with an antibody of theinvention and the other associated with a reference/comparator antibody)such that one of skill in the art would consider the difference betweenthe two values to be of little or no biological and/or statisticalsignificance within the context of the biological characteristicmeasured by said values (e.g., Kd values). The difference between saidtwo values is preferably less than about 50%, preferably less than about40%, preferably less than about 30%, preferably less than about 20%,preferably less than about 10% as a function of the value for thereference/comparator antibody.

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). Desirably the Kd is 1×10⁻⁷, 1×10⁻⁸, 5×10⁻⁸,1×10⁻⁹, 3×10⁻⁹, 5×10⁻⁹, or even 1×10⁻¹⁰ or stronger. Affinity can bemeasured by common methods known in the art, including those describedherein. Low-affinity antibodies generally bind antigen slowly and tendto dissociate readily, whereas high-affinity antibodies generally bindantigen faster and tend to remain bound longer. A variety of methods ofmeasuring binding affinity are known in the art, any of which can beused for purposes of the present invention. Specific illustrativeembodiments are described in the following.

In one embodiment, the “Kd” or “Kd value” according to this invention ismeasured by a radiolabeled antigen binding assay (RIA) performed withthe Fab version of an antibody of interest and its antigen as describedby the following assay that measures solution binding affinity of Fabsfor antigen by equilibrating Fab with a minimal concentration of(¹²⁵I)-labeled antigen in the presence of a titration series ofunlabeled antigen, then capturing bound antigen with an anti-Fabantibody-coated plate (Chen, et al., (1999) J. Mol. Biol. 293:865-881).To establish conditions for the assay, microtiter plates (Dynex) arecoated overnight with 5 μg/ml of a capturing anti-Fab antibody (CappelLabs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with2% (w/v) bovine serum albumin in PBS for two to five hours at roomtemperature (approximately 23° C.). In a non-adsorbant plate (Nunc#269620), 100 pM or 26 pM [¹²⁵I]-antigen are mixed with serial dilutionsof a Fab of interest (e.g., consistent with assessment of an anti-VEGFantibody, Fab-12, in Presta et al., (1997) Cancer Res. 57:4593-4599).The Fab of interest is then incubated overnight; however, the incubationmay continue for a longer period (e.g., 65 hours) to insure thatequilibrium is reached. Thereafter, the mixtures are transferred to thecapture plate for incubation at room temperature (e.g., for one hour).The solution is then removed and the plate washed eight times with 0.1%Tween-20 in PBS. When the plates have dried, 150 l/well of scintillant(MicroScint-20; Packard) is added, and the plates are counted on aTopcount gamma counter (Packard) for ten minutes. Concentrations of eachFab that give less than or equal to 20% of maximal binding are chosenfor use in competitive binding assays. According to another embodimentthe Kd or Kd value is measured by using surface plasmon resonance assaysusing a BIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway,N.J.) at 25° C. with immobilized antigen CM5 chips at ˜10 response units(RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcoreInc.) are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, into 5 g/ml (˜0.2 μM) before injection at a flow rate of 5μl/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1M ethanolamine is injectedto block unreacted groups. For kinetics measurements, two-fold serialdilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05%Tween 20 (PBST) at 25° C. at a flow rate of approximately 25 μl/min. Insome embodiments, the following modifications are used for the surfacePlasmon resonance assay method: antibody is immobilized to CM5 biosensorchips to achieve approximately 400 RU, and for kinetic measurements,two-fold serial dilutions of target protein (e.g., FGFR3-IIIb or -IIIc)(starting from 67 nM) are injected in PBST buffer at 25° C. with a flowrate of about 30 ul/minute. Association rates (k_(on)) and dissociationrates (k_(off)) are calculated using a simple one-to-one Langmuirbinding model (BIAcore Evaluation Software version 3.2) by simultaneousfitting the association and dissociation sensorgram. The equilibriumdissociation constant (Kd) is calculated as the ratio k_(off)/k_(on).See, e.g., Chen, Y., et al., (1999) J. Mol. Biol. 293:865-881. If theon-rate exceeds 10⁶ M⁻¹ S⁻¹ by the surface plasmon resonance assayabove, then the on-rate can be determined by using a fluorescentquenching technique that measures the increase or decrease influorescence emission intensity (excitation=295 nm; emission=340 nm, 16nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form) inPBS, pH 7.2, in the presence of increasing concentrations of antigen asmeasured in a spectrometer, such as a stop-flow equipped spectrophometer(Aviv Instruments) or a 8000-series SLM-Aminco spectrophotometer(ThermoSpectronic) with a stir red cuvette.

An “on-rate” or “rate of association” or “association rate” or “k_(on)”according to this invention can also be determined with the same surfaceplasmon resonance technique described above using a BIAcore™-2000 or aBIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C. withimmobilized antigen CM5 chips at ˜10 response units (RU). Briefly,carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) areactivated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, into 5 μg/ml (˜0.2 uM) before injection at a flow rate of 5μl/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1M ethanolamine is injectedto block unreacted groups. For kinetics measurements, two-fold serialdilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05%Tween 20 (PBST) at 25° C. at a flow rate of approximately 25 μl/min. Insome embodiments, the following modifications are used for the surfacePlasmon resonance assay method: antibody is immobilized to CM5 biosensorchips to achieve approximately 400 RU, and for kinetic measurements,two-fold serial dilutions of target protein (e.g., FGFR3-IIIb or -IIIc)(starting from 67 nM) are injected in PBST buffer at 25° C. with a flowrate of about 30 ul/minute. Association rates (k_(on)) and dissociationrates (k_(off)) are calculated using a simple one-to-one Langmuirbinding model (BIAcore Evaluation Software version 3.2) by simultaneousfitting the association and dissociation sensorgram. The equilibriumdissociation constant (Kd) was calculated as the ratio k_(off)/k_(on).See, e.g., Chen, Y., et al., (1999) J. Mol. Biol. 293:865-881. However,if the on-rate exceeds 10⁶ M⁻¹ S⁻¹ by the surface plasmon resonanceassay above, then the on-rate is preferably determined by using afluorescent quenching technique that measures the increase or decreasein fluorescence emission intensity (excitation=295 nm; emission=340 nm,16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form)in PBS, pH 7.2, in the presence of increasing concentrations of antigenas measured in a spectrometer, such as a stop-flow equippedspectrophometer (Aviv Instruments) or a 8000-series SLM-Amincospectrophotometer (ThermoSpectronic) with a stir red cuvette.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a phage vector. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)can be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors” (or simply, “recombinantvectors”). In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” may be used interchangeably as theplasmid is the most commonly used form of vector.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase, or by a syntheticreaction. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and their analogs. If present, modification tothe nucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter synthesis, such as by conjugation with a label. Other types ofmodifications include, for example, “caps,” substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelators (e.g., metals, radioactivemetals, boron, oxidative metals, etc.), those containing alkylators,those with modified linkages (e.g., alpha anomeric nucleic acids, etc.),as well as unmodified forms of the polynucleotide(s). Further, any ofthe hydroxyl groups ordinarily present in the sugars may be replaced,for example, by phosphonate groups, phosphate groups, protected bystandard protecting groups, or activated to prepare additional linkagesto additional nucleotides, or may be conjugated to solid or semi-solidsupports. The 5′ and 3′ terminal OH can be phosphorylated or substitutedwith amines or organic capping group moieties of from 1 to 20 carbonatoms. Other hydroxyls may also be derivatized to standard protectinggroups. Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including, forexample, 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or 2′-azido-ribose,carbocyclic sugar analogs, alpha-anomeric sugars, epimeric sugars suchas arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars,sedoheptuloses, acyclic analogs and a basic nucleoside analogs such asmethyl riboside. One or more phosphodiester linkages may be replaced byalternative linking groups. These alternative linking groups include,but are not limited to, embodiments wherein phosphate is replaced byP(O)S (“thioate”), P(S)S (“dithioate”), (O)NR₂ (“amidate”), P(O)R,P(O)OR′, CO or CH₂ (“formacetal”), in which each R or R′ isindependently H or substituted or unsubstituted alkyl (1-20 C)optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl,cycloalkenyl or araldyl. Not all linkages in a polynucleotide need beidentical. The preceding description applies to all polynucleotidesreferred to herein, including RNA and DNA.

“Oligonucleotide,” as used herein, generally refers to short, generallysingle stranded, generally synthetic polynucleotides that are generally,but not necessarily, less than about 200 nucleotides in length. Theterms “oligonucleotide” and “polynucleotide” are not mutually exclusive.The description above for polynucleotides is equally and fullyapplicable to oligonucleotides.

“Percent (%) amino acid sequence identity” with respect to a peptide orpolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the specific peptide or polypeptide sequence, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN orMegalign (DNASTAR) software. Those skilled in the art can determineappropriate parameters for measuring alignment, including any algorithmsneeded to achieve maximal alignment over the full length of thesequences being compared. For purposes herein, however, % amino acidsequence identity values are generated using the sequence comparisoncomputer program ALIGN-2, wherein the complete source code for theALIGN-2 program is provided in Table A below. The ALIGN-2 sequencecomparison computer program was authored by Genentech, Inc. and thesource code has been filed with user documentation in the U.S. CopyrightOffice, Washington D.C., 20559, where it is registered under U.S.Copyright Registration No. TXU510087. The ALIGN-2 program is publiclyavailable through Genentech, Inc., South San Francisco, Calif. or may becompiled from the source code provided in, e.g., WO2007/001851. TheALIGN-2 program should be compiled for use on a UNIX operating system,preferably digital UNIX V4.0D. All sequence comparison parameters areset by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A.

In some embodiments, two or more amino acid sequences are at least 50%,60%, 70%, 80%, or 90% identical. In some embodiments, two or more aminoacid sequences are at least 95%, 97%, 98%, 99%, or even 100% identical.Unless specifically stated otherwise, all % amino acid sequence identityvalues used herein are obtained as described in the immediatelypreceding paragraph using the ALIGN-2 computer program.

The term “FGFR3,” as used herein, refers, unless specifically orcontextually indicated otherwise, to any native or variant (whethernative or synthetic) FGFR3 polypeptide (e.g., FGFR3-IIIb isoform orFGFR3-IIIc isoform). The term “native sequence” specifically encompassesnaturally occurring truncated forms (e.g., an extracellular domainsequence or a transmembrane subunit sequence), naturally occurringvariant forms (e.g., alternatively spliced forms) andnaturally-occurring allelic variants. The term “wild-type FGFR3”generally refers to a polypeptide comprising an amino acid sequence of anaturally occurring FGFR3 protein. The term “wild type FGFR3 sequence”generally refers to an amino acid sequence found in a naturallyoccurring FGFR3.

The term “FGFR3 ligand,” (interchangeably termed “FGF”) as used herein,refers, unless specifically or contextually indicated otherwise, to anynative or variant (whether native or synthetic) FGFR3 ligand (forexample, FGF1, FGF2, FGF4, FGF8, FGF9, FGF17, FGF18, FGF23) polypeptide.The term “native sequence” specifically encompasses naturally occurringtruncated forms (e.g., an extracellular domain sequence or atransmembrane subunit sequence), naturally occurring variant forms(e.g., alternatively spliced forms) and naturally-occurring allelicvariants. The term “wild-type FGFR3 ligand” generally refers to apolypeptide comprising an amino acid sequence of a naturally occurringFGFR3 ligand protein. The term “wild type FGFR3 ligand sequence”generally refers to an amino acid sequence found in a naturallyoccurring FGFR3 ligand.

“FGFR3 activation” refers to activation, or phosphorylation, of theFGFR3 receptor. Generally, FGFR3 activation results in signaltransduction (e.g. that caused by an intracellular kinase domain of aFGFR3 receptor phosphorylating tyrosine residues in FGFR3 or a substratepolypeptide). FGFR3 activation may be mediated by FGFR ligand binding toa FGFR3 receptor of interest. FGFR3 ligand (e.g., such as FGF1 or FGF9)binding to FGFR3 may activate a kinase domain of FGFR3 and therebyresult in phosphorylation of tyrosine residues in the FGFR3 and/orphosphorylation of tyrosine residues in additional substratepolypeptides(s).

The term “constitutive” as used herein, as for example applied toreceptor kinase activity, refers to continuous signaling activity of areceptor that is not dependent on the presence of a ligand or otheractivating molecules. Depending on the nature of the receptor, all ofthe activity may be constitutive or the activity of the receptor may befurther activated by the binding of other molecules (e. g. ligands).Cellular events that lead to activation of receptors are well knownamong those of ordinary skill in the art. For example, activation mayinclude oligomerization, e.g., dimerization, trimerization, etc., intohigher order receptor complexes. Complexes may comprise a single speciesof protein, i.e., a homomeric complex. Alternatively, complexes maycomprise at least two different protein species, i.e., a heteromericcomplex. Complex formation may be caused by, for example, overexpressionof normal or mutant forms of receptor on the surface of a cell. Complexformation may also be caused by a specific mutation or mutations in areceptor.

The term “ligand-independent” as used herein, as for example applied toreceptor signaling activity, refers to signaling activity that is notdependent on the presence of a ligand. A receptor havingligand-independent kinase activity will not necessarily preclude thebinding of ligand to that receptor to produce additional activation ofthe kinase activity.

The term “ligand-dependent” as used herein, as for example applied toreceptor signaling activity, refers to signaling activity that isdependent on the presence of a ligand.

The phrase “gene amplification” refers to a process by which multiplecopies of a gene or gene fragment are formed in a particular cell orcell line. The duplicated region (a stretch of amplified DNA) is oftenreferred to as “amplicon.” Usually, the amount of the messenger RNA(mRNA) produced, i.e., the level of gene expression, also increases inthe proportion of the number of copies made of the particular geneexpressed.

A “tyrosine kinase inhibitor” is a molecule which inhibits to someextent tyrosine kinase activity of a tyrosine kinase such as a FGFR3receptor.

A cancer or biological sample which “displays FGFR3 expression,amplification, or activation” is one which, in a diagnostic test,expresses (including overexpresses) FGFR3, has amplified FGFR3 gene,and/or otherwise demonstrates activation or phosphorylation of a FGFR3.

A cancer or biological sample which “displays FGFR3 activation” is onewhich, in a diagnostic test, demonstrates activation or phosphorylationof FGFR3. Such activation can be determined directly (e.g. by measuringFGFR3 phosphorylation by ELISA) or indirectly.

A cancer or biological sample which “displays constitutive FGFR3activation” is one which, in a diagnostic test, demonstratesconstitutive activation or phosphorylation of a FGFR3. Such activationcan be determined directly (e.g. by measuring c-FGFR3 phosphorylation byELISA) or indirectly.

A cancer or biological sample which “displays FGFR3 amplification” isone which, in a diagnostic test, has amplified FGFR3 gene.

A cancer or biological sample which “displays FGFR3 translocation” isone which, in a diagnostic test, has translocated FGFR3 gene. An exampleof a FGFR3 translocation is the t(4;14) translocation, which occurs insome multiple myeloma tumors.

A “phospho-ELISA assay” herein is an assay in which phosphorylation ofone or more FGFR3, substrate or downstream signaling molecules isevaluated in an enzyme-linked immunosorbent assay (ELISA) using areagent, usually an antibody, to detect phosphorylated FGFR3, substrate,or downstream signaling molecule. In some embodiments, an antibody whichdetects phosphorylated FGFR3 or pMAPK is used. The assay may beperformed on cell lysates, preferably from fresh or frozen biologicalsamples.

A cancer or biological sample which “displays ligand-independent FGFR3activation” is one which, in a diagnostic test, demonstratesligand-independent activation or phosphorylation of a FGFR3. Suchactivation can be determined directly (e.g. by measuring FGFR3phosphorylation by ELISA) or indirectly.

A cancer or biological sample which “displays ligand-dependent FGFR3activation” is one which, in a diagnostic test, demonstratesligand-dependent activation or phosphorylation of a FGFR3. Suchactivation can be determined directly (e.g. by measuring FGFR3phosphorylation by ELISA) or indirectly.

A cancer or biological sample which “displays ligand-independent FGFR3activation” is one which, in a diagnostic test, demonstratesligand-independent activation or phosphorylation of a FGFR3. Suchactivation can be determined directly (e.g. by measuring FGFR3phosphorylation by ELISA) or indirectly.

A cancer cell with “FGFR3 overexpression or amplification” is one whichhas significantly higher levels of a FGFR3 protein or gene compared to anoncancerous cell of the same tissue type. Such overexpression may becaused by gene amplification or by increased transcription ortranslation. FGFR3 overexpression or amplification may be determined ina diagnostic or prognostic assay by evaluating increased levels of theFGFR3 protein present on the surface of a cell (e.g. via animmunohistochemistry assay; IHC). Alternatively, or additionally, onemay measure levels of FGFR3-encoding nucleic acid in the cell, e.g. viafluorescent in situ hybridization (FISH; see WO98/45479 publishedOctober, 1998), southern blotting, or polymerase chain reaction (PCR)techniques, such as quantitative real time PCR (qRT-PCR). Aside from theabove assays, various in vivo assays are available to the skilledpractitioner. For example, one may expose cells within the body of thepatient to an antibody which is optionally labeled with a detectablelabel, e.g. a radioactive isotope, and binding of the antibody to cellsin the patient can be evaluated, e.g. by external scanning forradioactivity or by analyzing a biopsy taken from a patient previouslyexposed to the antibody.

The term “mutation”, as used herein, means a difference in the aminoacid or nucleic acid sequence of a particular protein or nucleic acid(gene, RNA) relative to the wild-type protein or nucleic acid,respectively. A mutated protein or nucleic acid can be expressed from orfound on one allele (heterozygous) or both alleles (homozygous) of agene, and may be somatic or germ line. In the instant invention,mutations are generally somatic. Mutations include sequencerearrangements such as insertions, deletions, and point mutations(including single nucleotide/amino acid polymorphisms).

To “inhibit” is to decrease or reduce an activity, function, and/oramount as compared to a reference.

An agent possesses “agonist activity or function” when an agent mimicsat least one of the functional activities of a polypeptide of interest(e.g., FGFR ligand, such as FGF 1 or FGF9).

An “agonist antibody”, as used herein, is an antibody which mimics atleast one of the functional activities of a polypeptide of interest(e.g., FGFR ligand, such as FGF1 or FGF9).

Protein “expression” refers to conversion of the information encoded ina gene into messenger RNA (mRNA) and then to the protein.

Herein, a sample or cell that “expresses” a protein of interest (such asa FGF receptor or FGF receptor ligand) is one in which mRNA encoding theprotein, or the protein, including fragments thereof, is determined tobe present in the sample or cell.

An “immunoconjugate” (interchangeably referred to as “antibody-drugconjugate,” or “ADC”) means an antibody conjugated to one or morecytotoxic agents, such as a chemotherapeutic agent, a drug, a growthinhibitory agent, a toxin (e.g., a protein toxin, an enzymaticallyactive toxin of bacterial, fungal, plant, or animal origin, or fragmentsthereof), or a radioactive isotope (i.e., a radioconjugate).

The term “Fc region”, as used herein, generally refers to a dimercomplex comprising the C-terminal polypeptide sequences of animmunoglobulin heavy chain, wherein a C-terminal polypeptide sequence isthat which is obtainable by papain digestion of an intact antibody. TheFc region may comprise native or variant Fc sequences. Although theboundaries of the Fc sequence of an immunoglobulin heavy chain mightvary, the human IgG heavy chain Fc sequence is usually defined tostretch from an amino acid residue at about position Cys226, or fromabout position Pro230, to the carboxyl terminus of the Fc sequence. TheFc sequence of an immunoglobulin generally comprises two constantdomains, a CH2 domain and a CH3 domain, and optionally comprises a CH4domain. The C-terminal lysine (residue 447 according to the EU numberingsystem) of the Fc region may be removed, for example, duringpurification of the antibody or by recombinant engineering of thenucleic acid encoding the antibody. Accordingly, a compositioncomprising an antibody having an Fc region according to this inventioncan comprise an antibody with K447, with all K447 removed, or a mixtureof antibodies with and without the K447 residue.

By “Fc polypeptide” herein is meant one of the polypeptides that make upan Fc region. An Fc polypeptide may be obtained from any suitableimmunoglobulin, such as IgG₁, IgG₂, IgG₃, or IgG₄ subtypes, IgA, IgE,IgD or IgM. In some embodiments, an Fc polypeptide comprises part or allof a wild type hinge sequence (generally at its N terminus). In someembodiments, an Fc polypeptide does not comprise a functional or wildtype hinge sequence.

A “blocking” antibody or an antibody “antagonist” is one which inhibitsor reduces biological activity of the antigen it binds. Preferredblocking antibodies or antagonist antibodies completely inhibit thebiological activity of the antigen.

A “naked antibody” is an antibody that is not conjugated to aheterologous molecule, such as a cytotoxic moiety or radiolabel.

An antibody having a “biological characteristic” of a designatedantibody is one which possesses one or more of the biologicalcharacteristics of that antibody which distinguish it from otherantibodies that bind to the same antigen.

In order to screen for antibodies which bind to an epitope on an antigenbound by an antibody of interest, a routine cross-blocking assay such asthat described in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.

To increase the half-life of the antibodies or polypeptide containingthe amino acid sequences of this invention, one can attach a salvagereceptor binding epitope to the antibody (especially an antibodyfragment), as described, e.g., in U.S. Pat. No. 5,739,277. For example,a nucleic acid molecule encoding the salvage receptor binding epitopecan be linked in frame to a nucleic acid encoding a polypeptide sequenceof this invention so that the fusion protein expressed by the engineerednucleic acid molecule comprises the salvage receptor binding epitope anda polypeptide sequence of this invention. As used herein, the term“salvage receptor binding epitope” refers to an epitope of the Fc regionof an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, or IgG₄) that is responsiblefor increasing the in vivo serum half-life of the IgG molecule (e.g.,Ghetie et al., Ann. Rev. Immunol. 18:739-766 (2000), Table 1).Antibodies with substitutions in an Fc region thereof and increasedserum half-lives are also described in WO00/42072, WO 02/060919; Shieldset al., J. Biol. Chem. 276:6591-6604 (2001); Hinton, J. Biol. Chem.279:6213-6216 (2004)). In another embodiment, the serum half-life canalso be increased, for example, by attaching other polypeptidesequences. For example, antibodies or other polypeptides useful in themethods of the invention can be attached to serum albumin or a portionof serum albumin that binds to the FcRn receptor or a serum albuminbinding peptide so that serum albumin binds to the antibody orpolypeptide, e.g., such polypeptide sequences are disclosed inWO01/45746. In one preferred embodiment, the serum albumin peptide to beattached comprises an amino acid sequence of DICLPRWGCLW (SEQ ID NO:183). In another embodiment, the half-life of a Fab is increased bythese methods. See also, Dennis et al. J. Biol. Chem. 277:35035-35043(2002) for serum albumin binding peptide sequences.

By “fragment” is meant a portion of a polypeptide or nucleic acidmolecule that contains, preferably, at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, or more of the entire length of the referencenucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30,40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, or morenucleotides or 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160,180, 190, 200 amino acids or more.

The phrase “little to no agonist function” with respect to an antibodyof the invention, as used herein, means the antibody does not elicit abiologically meaningful amount of agonist activity, e.g., uponadministration to a subject. As would be understood in the art, amountof an activity may be determined quantitatively or qualitatively, solong as a comparison between an antibody of the invention and areference counterpart can be done. The activity can be measured ordetected according to any assay or technique known in the art,including, e.g., those described herein. The amount of activity for anantibody of the invention and its reference counterpart can bedetermined in parallel or in separate runs. In some embodiments, abivalent antibody of the invention does not possess substantial agonistfunction.

The terms “apoptosis” and “apoptotic activity” are used in a broad senseand refer to the orderly or controlled form of cell death in mammalsthat is typically accompanied by one or more characteristic cellchanges, including condensation of cytoplasm, loss of plasma membranemicrovilli, segmentation of the nucleus, degradation of chromosomal DNAor loss of mitochondrial function. This activity can be determined andmeasured using techniques known in the art, for instance, by cellviability assays, FACS analysis or DNA electrophoresis, and morespecifically by binding of annexin V, fragmentation of DNA, cellshrinkage, dilation of endoplasmatic reticulum, cell fragmentation,and/or formation of membrane vesicles (called apoptotic bodies).

The terms “antibody” and “immunoglobulin” are used interchangeably inthe broadest sense and include monoclonal antibodies (e.g., full lengthor intact monoclonal antibodies), polyclonal antibodies, multivalentantibodies, multispecific antibodies (e.g., bispecific antibodies solong as they exhibit the desired biological activity) and may alsoinclude certain antibody fragments (as described in greater detailherein). An antibody can be human, humanized, and/or affinity matured.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called complementarity-determining regions (CDRs) orhypervariable regions both in the light-chain and the heavy-chainvariable domains. The more highly conserved portions of variable domainsare called the framework (FR). The variable domains of native heavy andlight chains each comprise four FR regions, largely adopting a β-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the β-sheet structure. The CDRs in eachchain are held together in close proximity by the FR regions and, withthe CDRs from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al., Sequences ofProteins of Immunological Interest, Fifth Edition, National Institute ofHealth, Bethesda, Md. (1991)). The constant domains are not involveddirectly in binding an antibody to an antigen, but exhibit variouseffector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. In a two-chain Fv species, thisregion consists of a dimer of one heavy- and one light-chain variabledomain in tight, non-covalent association. In a single-chain Fv species,one heavy- and one light-chain variable domain can be covalently linkedby a flexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (x) and lambda (k), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these can be further divided into subclasses(isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. Theheavy-chain constant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known. “Antibody fragments” comprise only aportion of an intact antibody, wherein the portion preferably retains atleast one, preferably most or all, of the functions normally associatedwith that portion when present in an intact antibody. Examples ofantibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments;diabodies; linear antibodies; single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments. In oneembodiment, an antibody fragment comprises an antigen binding site ofthe intact antibody and thus retains the ability to bind antigen. Inanother embodiment, an antibody fragment, for example one that comprisesthe Fc region, retains at least one of the biological functions normallyassociated with the Fc region when present in an intact antibody, suchas FcRn binding, antibody half life modulation, ADCC function andcomplement binding. In one embodiment, an antibody fragment is amonovalent antibody that has an in vivo half life substantially similarto an intact antibody. For e.g., such an antibody fragment may compriseon antigen binding arm linked to an Fc sequence capable of conferring invivo stability to the fragment.

The term “hypervariable region,” “HVR,” or “HV,” when used herein refersto the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six hypervariable regions; three in the VH (H1, H2, H3), andthree in the VL (L1, L2, L3). A number of hypervariable regiondelineations are in use and are encompassed herein. The KabatComplementarity Determining Regions (CDRs) are based on sequencevariability and are the most commonly used (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Chothia refersinstead to the location of the structural loops (Chothia and Lesk, J.Mol. Biol. 196:901-917 (1987)). The AbM hypervariable regions representa compromise between the Kabat CDRs and Chothia structural loops, andare used by Oxford Molecular's AbM antibody modeling software. The“contact” hypervariable regions are based on an analysis of theavailable complex crystal structures. The residues from each of thesehypervariable regions are noted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1H31-H35B H26-H35B H26-H32 H30-H35B (Kabat Numbering) H1 H31-H35 H26-H35H26-H32 H30-H35 (Chothia Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101Hypervariable regions may comprise “extended hypervariable regions” asfollows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 (L3) in theVL and 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3)in the VH. The variable domain residues are numbered according to Kabatet al., supra for each of these definitions.

“Framework” or “FR” residues are those variable domain residues otherthan the hypervariable region residues as herein defined.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally will also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the followingreview articles and references cited therein: Vaswani and Hamilton, Ann.Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc.Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech.5:428-433 (1994).

“Chimeric” antibodies (immunoglobulins) have a portion of the heavyand/or light chain identical with or homologous to correspondingsequences in antibodies derived from a particular species or belongingto a particular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).Humanized antibody as used herein is a subset of chimeric antibodies.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VLdomains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the scFv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding. For a review of scFvsee Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

An “antigen” is a predetermined antigen to which an antibody canselectively bind. The target antigen may be polypeptide, carbohydrate,nucleic acid, lipid, hapten or other naturally occurring or syntheticcompound. Preferably, the target antigen is a polypeptide.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993).

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues.

An “affinity matured” antibody is one with one or more alterations inone or more CDRs thereof which result in an improvement in the affinityof the antibody for antigen, compared to a parent antibody which doesnot possess those alteration(s). Preferred affinity matured antibodieswill have nanomolar or even picomolar affinities for the target antigen.Affinity matured antibodies are produced by procedures known in the art.Marks et al. Bio/Technology 10:779-783 (1992) describes affinitymaturation by VH and VL domain shuffling. Random mutagenesis of CDRand/or framework residues is described by: Barbas et al., Proc Nat.Acad. Sci, USA 91:3809-3813 (1994); Schier et al., Gene 169:147-155(1995); Yelton et al., J. Immunol. 155:1994-2004 (1995); Jackson et al.,J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol.226:889-896 (1992).

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: C1q bindingand complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cellactivation.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g., Natural Killer (NK) cells,neutrophils, and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. The antibodies “arm” the cytotoxiccells and are absolutely required for such killing. The primary cellsfor mediating ADCC, NK cells, express FcγRIII only, whereas monocytesexpress FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cellsis summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev.Immunol 9:457-92 (1991). To assess ADCC activity of a molecule ofinterest, an in vitro ADCC assay, such as that described in U.S. Pat.No. 5,500,362 or 5,821,337 or Presta U.S. Pat. No. 6,737,056 may beperformed. Useful effector cells for such assays include peripheralblood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g., in a animal model such as thatdisclosed in Clynes et al., PNAS (USA) 95:652-656 (1998).

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Examples of human leukocyteswhich mediate ADCC include peripheral blood mononuclear cells (PBMC),natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils;with PBMCs and NK cells being preferred. The effector cells may beisolated from a native source, e.g., from blood.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. The preferred FcR is a native sequence human FcR.Moreover, a preferred FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof these receptors. FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domain.Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. (see review M. inDaëron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed inRavetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al.,Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med.126:330-41 (1995). Other FcRs, including those to be identified in thefuture, are encompassed by the term “FcR” herein. The term also includesthe neonatal receptor, FcRn, which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)) and regulates homeostasis ofimmunoglobulins. WO 00/42072 (Presta) describes antibody variants withimproved or diminished binding to FcRs. The content of that patentpublication is specifically incorporated herein by reference. See, also,Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).

Methods of measuring binding to FcRn are known (see, e.g., Ghetie 1997,Hinton 2004). Binding to human FcRn in vivo and serum half life of humanFcRn high affinity binding polypeptides can be assayed, e.g, intransgenic mice or transfected human cell lines expressing human FcRn,or in primates administered with the Fc variant polypeptides.

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass)which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g., as described in Gazzano-Santoro et al.,J. Immunol. Methods 202:163 (1996), may be performed.

Polypeptide variants with altered Fc region amino acid sequences andincreased or decreased C1q binding capability are described in U.S. Pat.No. 6,194,551B1 and WO 99/51642. The contents of those patentpublications are specifically incorporated herein by reference. See,also, Idusogie et al., J. Immunol. 164:4178-4184 (2000).

The term “Fc region-comprising polypeptide” refers to a polypeptide,such as an antibody or immunoadhesin, which comprises an Fc region. TheC-terminal lysine (residue 447 according to the EU numbering system) ofthe Fc region may be removed, for example, during purification of thepolypeptide or by recombinant engineering the nucleic acid encoding thepolypeptide. Accordingly, a composition comprising a polypeptide havingan Fc region according to this invention can comprise polypeptides withK447, with all K447 removed, or a mixture of polypeptides with andwithout the K447 residue.

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a VL or VH framework derived froma human immunoglobulin framework, or from a human consensus framework.An acceptor human framework “derived from” a human immunoglobulinframework or human consensus framework may comprise the same amino acidsequence thereof, or may contain pre-existing amino acid sequencechanges. Where pre-existing amino acid changes are present, preferablyno more than 5 and preferably 4 or less, or 3 or less, pre-existingamino acid changes are present. Where pre-existing amino acid changesare present in a VH, preferably those changes are only at three, two, orone of positions 71H, 73H, and 78H; for instance, the amino acidresidues at those positions may be 71A, 73T, and/or 78A. In oneembodiment, the VL acceptor human framework is identical in sequence tothe VL human immunoglobulin framework sequence or human consensusframework sequence.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residue in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al. In one embodiment, for the VL, the subgroup is subgroupkappa I as in Kabat et al. In one embodiment, for the VH, the subgroupis subgroup III as in Kabat et al.

A “VH subgroup III consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable heavy subgroup III ofKabat et al. In one embodiment, the VH subgroup III consensus frameworkamino acid sequence comprises at least a portion or all of each of thefollowing sequences: EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:184)-H1-WVRQAPGKGLEWV (SEQ ID NO: 185)-H2-RFTISRDNSKNTLYLQMNSLRAEDTAVYYC(SEQ ID NO: 186)-H3-WGQGTLVTVS S (SEQ ID NO:187).

A “VL subgroup I consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable light kappa subgroupI of Kabat et al. In one embodiment, the VH subgroup I consensusframework amino acid sequence comprises at least a portion or all ofeach of the following sequences:

(SEQ ID NO: 188) DIQMTQSPSSLSASVGDRVTITC -L1- (SEQ ID NO: 189)WYQQKPGKAPKLLIY -L2- (SEQ ID NO: 190) GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC -L3- (SEQ ID NO: 191) FGQGTKVEIK.

As used herein, “antibody mutant” or “antibody variant” refers to anamino acid sequence variant of an antibody wherein one or more of theamino acid residues of the species-dependent antibody have beenmodified. Such mutants necessarily have less than 100% sequence identityor similarity with the species-dependent antibody. In one embodiment,the antibody mutant will have an amino acid sequence having at least 75%amino acid sequence identity or similarity with the amino acid sequenceof either the heavy or light chain variable domain of thespecies-dependent antibody, more preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, and mostpreferably at least 95%. Identity or similarity with respect to thissequence is defined herein as the percentage of amino acid residues inthe candidate sequence that are identical (i.e same residue) or similar(i.e. amino acid residue from the same group based on common side-chainproperties, see below) with the species-dependent antibody residues,after aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent sequence identity. None of N-terminal,C-terminal, or internal extensions, deletions, or insertions into theantibody sequence outside of the variable domain shall be construed asaffecting sequence identity or similarity

A “disorder” or “disease” is any condition that would benefit fromtreatment with a substance/molecule or method of the invention. Thisincludes chronic and acute disorders or diseases including thosepathological conditions which predispose the mammal to the disorder inquestion. Non-limiting examples of disorders to be treated hereininclude malignant and benign tumors; carcinoma, blastoma, and sarcoma.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadyhaving a benign, pre-cancerous, or non-metastatic tumor as well as thosein which the occurrence or recurrence of cancer is to be prevented.

The term “therapeutically effective amount” refers to an amount of atherapeutic agent to treat or prevent a disease or disorder in a mammal.In the case of cancers, the therapeutically effective amount of thetherapeutic agent may reduce the number of cancer cells; reduce theprimary tumor size; inhibit (i.e., slow to some extent and preferablystop) cancer cell infiltration into peripheral organs; inhibit (i.e.,slow to some extent and preferably stop) tumor metastasis; inhibit, tosome extent, tumor growth; and/or relieve to some extent one or more ofthe symptoms associated with the disorder. To the extent the drug mayprevent growth and/or kill existing cancer cells, it may be cytostaticand/or cytotoxic. For cancer therapy, efficacy in vivo can, for example,be measured by assessing the duration of survival, time to diseaseprogression (TTP), the response rates (RR), duration of response, and/orquality of life.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Included in this definition are benign andmalignant cancers. By “early stage cancer” or “early stage tumor” ismeant a cancer that is not invasive or metastatic or is classified as aStage 0, I, or II cancer. Examples of cancer include, but are notlimited to, carcinoma, lymphoma, blastoma (including medulloblastoma andretinoblastoma), sarcoma (including liposarcoma and synovial cellsarcoma), neuroendocrine tumors (including carcinoid tumors, gastrinoma,and islet cell cancer), mesothelioma, schwannoma (including acousticneuroma), meningioma, adenocarcinoma, melanoma, and leukemia or lymphoidmalignancies. More particular examples of such cancers include squamouscell cancer (e.g. epithelial squamous cell cancer), lung cancerincluding small-cell lung cancer (SCLC), non-small cell lung cancer(NSCLC), adenocarcinoma of the lung and squamous carcinoma of the lung,cancer of the peritoneum, hepatocellular cancer, gastric or stomachcancer including gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer (including metastatic breast cancer),colon cancer, rectal cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, prostatecancer, vulval cancer, thyroid cancer, hepatic carcinoma, analcarcinoma, penile carcinoma, testicular cancer, esophagael cancer,tumors of the biliary tract, as well as head and neck cancer andmultiple myeloma.

The term “pre-cancerous” refers to a condition or a growth thattypically precedes or develops into a cancer. A “pre-cancerous” growthwill have cells that are characterized by abnormal cell cycleregulation, proliferation, or differentiation, which can be determinedby markers of cell cycle regulation, cellular proliferation, ordifferentiation.

By “dysplasia” is meant any abnormal growth or development of tissue,organ, or cells. Preferably, the dysplasia is high grade orprecancerous.

By “metastasis” is meant the spread of cancer from its primary site toother places in the body. Cancer cells can break away from a primarytumor, penetrate into lymphatic and blood vessels, circulate through thebloodstream, and grow in a distant focus (metastasize) in normal tissueselsewhere in the body. Metastasis can be local or distant. Metastasis isa sequential process, contingent on tumor cells breaking off from theprimary tumor, traveling through the bloodstream, and stopping at adistant site. At the new site, the cells establish a blood supply andcan grow to form a life-threatening mass.

Both stimulatory and inhibitory molecular pathways within the tumor cellregulate this behavior, and interactions between the tumor cell and hostcells in the distant site are also significant.

By “non-metastatic” is meant a cancer that is benign or that remains atthe primary site and has not penetrated into the lymphatic or bloodvessel system or to tissues other than the primary site. Generally, anon-metastatic cancer is any cancer that is a Stage 0, I, or II cancer,and occasionally a Stage III cancer.

By “primary tumor” or “primary cancer” is meant the original cancer andnot a metastatic lesion located in another tissue, organ, or location inthe subject's body.

By “benign tumor” or “benign cancer” is meant a tumor that remainslocalized at the site of origin and does not have the capacity toinfiltrate, invade, or metastasize to a distant site.

By “tumor burden” is meant the number of cancer cells, the size of atumor, or the amount of cancer in the body. Tumor burden is alsoreferred to as tumor load.

By “tumor number” is meant the number of tumors.

By “subject” is meant a mammal, including, but not limited to, a humanor non-human mammal, such as a bovine, equine, canine, ovine, or feline.Preferably, the subject is a human.

The term “anti-cancer therapy” refers to a therapy useful in treatingcancer. Examples of anti-cancer therapeutic agents include, but arelimited to, e.g., chemotherapeutic agents, growth inhibitory agents,cytotoxic agents, agents used in radiation therapy, anti-angiogenesisagents, apoptotic agents, anti-tubulin agents, and other agents to treatcancer, anti-CD20 antibodies, platelet derived growth factor inhibitors(e.g., Gleevec™ (Imatinib Mesylate)), a COX-2 inhibitor (e.g.,celecoxib), interferons, cytokines, antagonists (e.g., neutralizingantibodies) that bind to one or more of the following targets ErbB2,ErbB3, ErbB4, PDGFR-beta, BlyS, APRIL, BCMA or VEGF receptor(s),TRAIL/Apo2, and other bioactive and organic chemical agents, etc.Combinations thereof are also included in the invention.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g., I¹³¹,I¹²⁵, Y⁹⁰ and Re¹⁸⁶), chemotherapeutic agents, and toxins such asenzymatically active toxins of bacterial, fungal, plant or animalorigin, or fragments thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents include is achemical compound useful in the treatment of cancer. Examples ofchemotherapeutic agents include alkylating agents such as thiotepa andCYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan,improsulfan and piposulfan; aziridines such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB 1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e. g.,calicheamicin, especially calicheamicin gammalI and calicheamicinomegaI1 (see, e.g., Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994));dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antiobiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL®paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhône-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine;NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin;aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11)(including the treatment regimen of irinotecan with 5-FU andleucovorin); topoisomerase inhibitor RFS 2000; difluorometlhylornithine(DMFO); retinoids such as retinoic acid; capecitabine; combretastatin;VELCADE bortezomib; REVLIMID lenalidomide; leucovorin (LV); oxaliplatin,including the oxaliplatin treatment regimen (FOLFOX); inhibitors ofPKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva™)) and VEGF-A thatreduce cell proliferation and pharmaceutically acceptable salts, acidsor derivatives of any of the above.

Also included in this definition are anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogens andselective estrogen receptor modulators (SERMs), including, for example,tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andFARESTON-toremifene; aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE®megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole,RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole; andanti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide,and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleosidecytosine analog); antisense oligonucleotides, particularly those whichinhibit expression of genes in signaling pathways implicated in abherantcell proliferation, such as, for example, PKC-alpha, Raf and H-Ras;ribozymes such as a VEGF expression inhibitor (e.g., ANGIOZYME®ribozyme) and a HER2 expression inhibitor; vaccines such as gene therapyvaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, andVAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitor;ABARELIX® rmRH; Vinorelbine and Esperamicins (see U.S. Pat. No.4,675,187), and pharmaceutically acceptable salts, acids or derivativesof any of the above.

The term “prodrug” as used in this application refers to a precursor orderivative form of a pharmaceutically active substance that is lesscytotoxic to tumor cells compared to the parent drug and is capable ofbeing enzymatically activated or converted into the more active parentform. See, e.g., Wilman, “Prodrugs in Cancer Chemotherapy” BiochemicalSociety Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) andStella et al., “Prodrugs: A Chemical Approach to Targeted DrugDelivery,” Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267,Humana Press (1985). The prodrugs of this invention include, but are notlimited to, phosphate-containing prodrugs, thiophosphate-containingprodrugs, sulfate-containing prodrugs, peptide-containing prodrugs,D-amino acid-modified prodrugs, glycosylated prodrugs,β-lactam-containing prodrugs, optionally substitutedphenoxyacetamide-containing prodrugs or optionally substitutedphenylacetamide-containing prodrugs, 5-fluorocytosine and other5-fluorouridine prodrugs which can be converted into the more activecytotoxic free drug. Examples of cytotoxic drugs that can be derivatizedinto a prodrug form for use in this invention include, but are notlimited to, those chemotherapeutic agents described above.

By “radiation therapy” is meant the use of directed gamma rays or betarays to induce sufficient damage to a cell so as to limit its ability tofunction normally or to destroy the cell altogether. It will beappreciated that there will be many ways known in the art to determinethe dosage and duration of treatment. Typical treatments are given as aone time administration and typical dosages range from 10 to 200 units(Grays) per day.

A “biological sample” (interchangeably termed “sample” or “tissue orcell sample”) encompasses a variety of sample types obtained from anindividual and can be used in a diagnostic or monitoring assay. Thedefinition encompasses blood and other liquid samples of biologicalorigin, solid tissue samples such as a biopsy specimen or tissuecultures or cells derived therefrom, and the progeny thereof. Thedefinition also includes samples that have been manipulated in any wayafter their procurement, such as by treatment with reagents,solubilization, or enrichment for certain components, such as proteinsor polynucleotides, or embedding in a semi-solid or solid matrix forsectioning purposes. The term “biological sample” encompasses a clinicalsample, and also includes cells in culture, cell supernatants, celllysates, serum, plasma, biological fluid, and tissue samples. The sourceof the biological sample may be solid tissue as from a fresh, frozenand/or preserved organ or tissue sample or biopsy or aspirate; blood orany blood constituents; bodily fluids such as cerebral spinal fluid,amniotic fluid, peritoneal fluid, or interstitial fluid; cells from anytime in gestation or development of the individual. In some embodiments,the biological sample is obtained from a primary or metastatic tumor.The biological sample may contain compounds which are not naturallyintermixed with the tissue in nature such as preservatives,anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.

For the purposes herein a “section” of a tissue sample is meant a singlepart or piece of a tissue sample, e.g., a thin slice of tissue or cellscut from a tissue sample. It is understood that multiple sections oftissue samples may be taken and subjected to analysis according to thepresent invention. In some embodiments, the same section of tissuesample is analyzed at both morphological and molecular levels, or isanalyzed with respect to both protein and nucleic acid.

The word “label” when used herein refers to a compound or compositionwhich is conjugated or fused directly or indirectly to a reagent such asa nucleic acid probe or an antibody and facilitates detection of thereagent to which it is conjugated or fused. The label may itself bedetectable (e.g., radioisotope labels or fluorescent labels) or, in thecase of an enzymatic label, may catalyze chemical alteration of asubstrate compound or composition which is detectable.

Compositions and Methods of the Invention

This invention encompasses compositions, including pharmaceuticalcompositions, comprising an anti-FGFR3 antibody; and polynucleotidescomprising sequences encoding an anti-FGFR3 antibody. As used herein,compositions comprise one or more antibodies that bind to FGFR3, and/orone or more polynucleotides comprising sequences encoding one or moreantibodies that bind to FGFR3. These compositions may further comprisesuitable carriers, such as pharmaceutically acceptable excipientsincluding buffers, which are well known in the art.

The invention also encompasses isolated antibody and polynucleotideembodiments. The invention also encompasses substantially pure antibodyand polynucleotide embodiments.

The invention also encompasses method of treating a disorder, e.g.multiple myeloma or transitional stage carcinoma (e.g., invasivetransitional stage carcinoma) using an anti-FGFR3 antibody (as describedherein or as known in the art).

Compositions

The anti-FGFR3 antibodies of the invention are preferably monoclonal.Also encompassed within the scope of the invention are Fab, Fab′,Fab′-SH and F(ab′)₂ fragments of the anti-FGFR3 antibodies providedherein. These antibody fragments can be created by traditional means,such as enzymatic digestion, or may be generated by recombinanttechniques. Such antibody fragments may be chimeric or humanized. Thesefragments are useful for the diagnostic and therapeutic purposes setforth below.

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations that may be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies.

The anti-FGFR3 monoclonal antibodies of the invention can be made usingthe hybridoma method first described by Kohler et al., Nature, 256:495(1975), or may be made by recombinant DNA methods (U.S. Pat. No.4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized to elicit lymphocytes that produce or arecapable of producing antibodies that will specifically bind to theprotein used for immunization. Antibodies to FGFR3 may be raised inanimals by multiple subcutaneous (sc) or intraperitoneal (ip) injectionsof FGFR3 and an adjuvant. FGFR3 may be prepared using methods well-knownin the art, some of which are further described herein. For example,recombinant production of human and mouse FGFR3 is described below. Inone embodiment, animals are immunized with a FGFR3 fused to the Fcportion of an immunoglobulin heavy chain. In a preferred embodiment,animals are immunized with a FGFR3-IgG1 fusion protein. Animalsordinarily are immunized against immunogenic conjugates or derivativesof FGFR3 with monophosphoryl lipid A (MPL)/trehalose dicrynomycolate(TDM) (Ribi Immunochem. Research, Inc., Hamilton, Mont.) and thesolution is injected intradermally at multiple sites. Two weeks laterthe animals are boosted. 7 to 14 days later animals are bled and theserum is assayed for anti-FGFR3 titer. Animals are boosted until titerplateaus.

Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2 orX₆₃-Ag8-653 cells available from the American Type Culture Collection,Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma celllines also have been described for the production of human monoclonalantibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63(Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against FGFR3. Preferably,the binding specificity of monoclonal antibodies produced by hybridomacells is determined by immunoprecipitation or by an in vitro bindingassay, such as radioimmunoassay (RIA) or enzyme-linked immunoadsorbentassay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson et al., Anal. Biochem.,107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

The anti-FGFR3 antibodies of the invention can be made by usingcombinatorial libraries to screen for synthetic antibody clones with thedesired activity or activities. In principle, synthetic antibody clonesare selected by screening phage libraries containing phage that displayvarious fragments of antibody variable region (Fv) fused to phage coatprotein. Such phage libraries are panned by affinity chromatographyagainst the desired antigen. Clones expressing Fv fragments capable ofbinding to the desired antigen are adsorbed to the antigen and thusseparated from the non-binding clones in the library. The binding clonesare then eluted from the antigen, and can be further enriched byadditional cycles of antigen adsorption/elution. Any of the anti-FGFR3antibodies of the invention can be obtained by designing a suitableantigen screening procedure to select for the phage clone of interestfollowed by construction of a full length anti-FGFR3 antibody cloneusing the Fv sequences from the phage clone of interest and suitableconstant region (Fc) sequences described in Kabat et al., Sequences ofProteins of Immunological Interest, Fifth Edition, NIH Publication91-3242, Bethesda Md. (1991), vols. 1-3.

The antigen-binding domain of an antibody is formed from two variable(V) regions of about 110 amino acids, one each from the light (VL) andheavy (VH) chains, that both present three hypervariable loops orcomplementarity-determining regions (CDRs). Variable domains can bedisplayed functionally on phage, either as single-chain Fv (scFv)fragments, in which VH and VL are covalently linked through a short,flexible peptide, or as Fab fragments, in which they are each fused to aconstant domain and interact non-covalently, as described in Winter etal., Ann. Rev. Immunol., 12: 433-455 (1994). As used herein, scFvencoding phage clones and Fab encoding phage clones are collectivelyreferred to as “Fv phage clones” or “Fv clones”.

Repertoires of VH and VL genes can be separately cloned by polymerasechain reaction (PCR) and recombined randomly in phage libraries, whichcan then be searched for antigen-binding clones as described in Winteret al., Ann. Rev. Immunol., 12: 433-455 (1994). Libraries from immunizedsources provide high-affinity antibodies to the immunogen without therequirement of constructing hybridomas. Alternatively, the naiverepertoire can be cloned to provide a single source of human antibodiesto a wide range of non-self and also self antigens without anyimmunization as described by Griffiths et al., EMBO J, 12: 725-734(1993). Finally, naive libraries can also be made synthetically bycloning the unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).

Filamentous phage is used to display antibody fragments by fusion to theminor coat protein pIII. The antibody fragments can be displayed assingle chain Fv fragments, in which VH and VL domains are connected onthe same polypeptide chain by a flexible polypeptide spacer, e.g., asdescribed by Marks et al., J. Mol. Biol., 222: 581-597 (1991), or as Fabfragments, in which one chain is fused to pIII and the other is secretedinto the bacterial host cell periplasm where assembly of a Fab-coatprotein structure which becomes displayed on the phage surface bydisplacing some of the wild type coat proteins, e.g., as described inHoogenboom et al., Nucl. Acids Res., 19: 4133-4137 (1991).

In general, nucleic acids encoding antibody gene fragments are obtainedfrom immune cells harvested from humans or animals. If a library biasedin favor of anti-FGFR3 clones is desired, the individual is immunizedwith FGFR3 to generate an antibody response, and spleen cells and/orcirculating B cells other peripheral blood lymphocytes (PBLs) arerecovered for library construction. In a preferred embodiment, a humanantibody gene fragment library biased in favor of anti-FGFR3 clones isobtained by generating an anti-FGFR3 antibody response in transgenicmice carrying a functional human immunoglobulin gene array (and lackinga functional endogenous antibody production system) such that FGFR3immunization gives rise to B cells producing human antibodies againstFGFR3. The generation of human antibody-producing transgenic mice isdescribed below.

Additional enrichment for anti-FGFR3 reactive cell populations can beobtained by using a suitable screening procedure to isolate B cellsexpressing FGFR3-specific membrane bound antibody, e.g., by cellseparation with FGFR3 affinity chromatography or adsorption of cells tofluorochrome-labeled FGFR3 followed by flow-activated cell sorting(FACS).

Alternatively, the use of spleen cells and/or B cells or other PBLs froman unimmunized donor provides a better representation of the possibleantibody repertoire, and also permits the construction of an antibodylibrary using any animal (human or non-human) species in which FGFR3 isnot antigenic. For libraries incorporating in vitro antibody geneconstruction, stem cells are harvested from the individual to providenucleic acids encoding unrearranged antibody gene segments. The immunecells of interest can be obtained from a variety of animal species, suchas human, mouse, rat, lagomorpha, luprine, canine, feline, porcine,bovine, equine, and avian species, etc.

Nucleic acid encoding antibody variable gene segments (including VH andVL segments) are recovered from the cells of interest and amplified. Inthe case of rearranged VH and VL gene libraries, the desired DNA can beobtained by isolating genomic DNA or mRNA from lymphocytes followed bypolymerase chain reaction (PCR) with primers matching the 5′ and 3′ endsof rearranged VH and VL genes as described in Orlandi et al., Proc.Natl. Acad. Sci. (USA), 86: 3833-3837 (1989), thereby making diverse Vgene repertoires for expression. The V genes can be amplified from cDNAand genomic DNA, with back primers at the 5′ end of the exon encodingthe mature V-domain and forward primers based within the J-segment asdescribed in Orlandi et al. (1989) and in Ward et al., Nature, 341:544-546 (1989). However, for amplifying from cDNA, back primers can alsobe based in the leader exon as described in Jones et al., Biotechnol.,9: 88-89 (1991), and forward primers within the constant region asdescribed in Sastry et al., Proc. Natl. Acad. Sci. (USA), 86: 5728-5732(1989). To maximize complementarity, degeneracy can be incorporated inthe primers as described in Orlandi et al. (1989) or Sastry et al.(1989). Preferably, the library diversity is maximized by using PCRprimers targeted to each V-gene family in order to amplify all availableVH and VL arrangements present in the immune cell nucleic acid sample,e.g. as described in the method of Marks et al., J. Mol. Biol., 222:581-597 (1991) or as described in the method of Orum et al., NucleicAcids Res., 21: 4491-4498 (1993). For cloning of the amplified DNA intoexpression vectors, rare restriction sites can be introduced within thePCR primer as a tag at one end as described in Orlandi et al. (1989), orby further PCR amplification with a tagged primer as described inClackson et al., Nature, 352: 624-628 (1991).

Repertoires of synthetically rearranged V genes can be derived in vitrofrom V gene segments. Most of the human VH-gene segments have beencloned and sequenced (reported in Tomlinson et al., J. Mol. Biol., 227:776-798 (1992)), and mapped (reported in Matsuda et al., Nature Genet.,3: 88-94 (1993); these cloned segments (including all the majorconformations of the H1 and H2 loop) can be used to generate diverse VHgene repertoires with PCR primers encoding H3 loops of diverse sequenceand length as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). VH repertoires can also be made with all the sequencediversity focused in a long H3 loop of a single length as described inBarbas et al., Proc. Natl. Acad. Sci. USA, 89: 4457-4461 (1992). HumanVx and VX segments have been cloned and sequenced (reported in Williamsand Winter, Eur. J. Immunol., 23: 1456-1461 (1993)) and can be used tomake synthetic light chain repertoires. Synthetic V gene repertoires,based on a range of VH and VL folds, and L3 and H3 lengths, will encodeantibodies of considerable structural diversity. Following amplificationof V-gene encoding DNAs, germline V-gene segments can be rearranged invitro according to the methods of Hoogenboom and Winter, J. Mol. Biol.,227: 381-388 (1992).

Repertoires of antibody fragments can be constructed by combining VH andVL gene repertoires together in several ways. Each repertoire can becreated in different vectors, and the vectors recombined in vitro, e.g.,as described in Hogrefe et al., Gene, 128:119-126 (1993), or in vivo bycombinatorial infection, e.g., the loxP system described in Waterhouseet al., Nucl. Acids Res., 21:2265-2266 (1993). The in vivo recombinationapproach exploits the two-chain nature of Fab fragments to overcome thelimit on library size imposed by E. coli transformation efficiency.Naive VH and VL repertoires are cloned separately, one into a phagemidand the other into a phage vector. The two libraries are then combinedby phage infection of phagemid-containing bacteria so that each cellcontains a different combination and the library size is limited only bythe number of cells present (about 10¹² clones). Both vectors contain invivo recombination signals so that the VH and VL genes are recombinedonto a single replicon and are co-packaged into phage virions. Thesehuge libraries provide large numbers of diverse antibodies of goodaffinity (K_(d) ¹ of about 10⁻⁸ M).

Alternatively, the repertoires may be cloned sequentially into the samevector, e.g., as described in Barbas et al., Proc. Natl. Acad. Sci. USA,88:7978-7982 (1991), or assembled together by PCR and then cloned, e.g.as described in Clackson et al., Nature, 352: 624-628 (1991). PCRassembly can also be used to join VH and VL DNAs with DNA encoding aflexible peptide spacer to form single chain Fv (scFv) repertoires. Inyet another technique, “in cell PCR assembly” is used to combine VH andVL genes within lymphocytes by PCR and then clone repertoires of linkedgenes as described in Embleton et al., Nucl. Acids Res., 20:3831-3837(1992).

The antibodies produced by naive libraries (either natural or synthetic)can be of moderate affinity (K_(d) ¹ of about 10⁶ to 10⁷ M⁻¹), butaffinity maturation can also be mimicked in vitro by constructing andreselecting from secondary libraries as described in Winter et al.(1994), supra. For example, mutations can be introduced at random invitro by using error-prone polymerase (reported in Leung et al.,Technique, 1:11-15 (1989)) in the method of Hawkins et al., J. Mol.Biol., 226: 889-896 (1992) or in the method of Gram et al., Proc. Natl.Acad. Sci USA, 89: 3576-3580 (1992). Additionally, affinity maturationcan be performed by randomly mutating one or more CDRs, e.g. using PCRwith primers carrying random sequence spanning the CDR of interest, inselected individual Fv clones and screening for higher affinity clones.WO 96/07754 (published 14 Mar. 1996) described a method for inducingmutagenesis in a complementarity determining region of an immunoglobulinlight chain to create a library of light chain genes. Another effectiveapproach is to recombine the VH or VL domains selected by phage displaywith repertoires of naturally occurring V domain variants obtained fromunimmunized donors and screen for higher affinity in several rounds ofchain reshuffling as described in Marks et al., Biotechnol., 10:779-783(1992). This technique allows the production of antibodies and antibodyfragments with affinities in the 10⁻⁹ M range.

FGFR3 nucleic acid and amino acid sequences are known in the art.Nucleic acid sequence encoding the FGFR3 can be designed using the aminoacid sequence of the desired region of FGFR3. As is well-known in theart, there are two major splice isoforms of FGFR3, FGFR3 IIIb and FGFR3IIIc. FGFR3 sequences are well-known in the art and may include thesequence of UniProKB/Swiss-Prot accession number P22607 (FGFR3 IIIc) orP22607_2 (FGFR3 IIIb). FGFR3 mutations have been identified and arewell-known in the art and include the following mutations (withreference to the sequences shown in UniProKB/Swiss-Prot accession numberP22607 (FGFR3 IIIc) or P226072 (FGFR3 IIIb):

FGFR3-IIIb FGFR3 IIIc R248C R248C S249C S249C G372C G370C Y375C Y373CG382R G380R K652E K650E

Nucleic acids encoding FGFR3 can be prepared by a variety of methodsknown in the art. These methods include, but are not limited to,chemical synthesis by any of the methods described in Engels et al.,Agnew. Chem. Int. Ed. Engl., 28: 716-734 (1989), such as the triester,phosphite, phosphoramidite and H-phosphonate methods. In one embodiment,codons preferred by the expression host cell are used in the design ofthe FGFR3 encoding DNA. Alternatively, DNA encoding the FGFR3 can beisolated from a genomic or cDNA library.

Following construction of the DNA molecule encoding the FGFR3, the DNAmolecule is operably linked to an expression control sequence in anexpression vector, such as a plasmid, wherein the control sequence isrecognized by a host cell transformed with the vector. In general,plasmid vectors contain replication and control sequences which arederived from species compatible with the host cell. The vectorordinarily carries a replication site, as well as sequences which encodeproteins that are capable of providing phenotypic selection intransformed cells. Suitable vectors for expression in prokaryotic andeukaryotic host cells are known in the art and some are furtherdescribed herein. Eukaryotic organisms, such as yeasts, or cells derivedfrom multicellular organisms, such as mammals, may be used.

Optionally, the DNA encoding the FGFR3 is operably linked to a secretoryleader sequence resulting in secretion of the expression product by thehost cell into the culture medium. Examples of secretory leadersequences include stII, ecotin, lamB, herpes GD, lpp, alkalinephosphatase, invertase, and alpha factor. Also suitable for use hereinis the 36 amino acid leader sequence of protein A (Abrahmsen et al.,EMBO J., 4: 3901 (1985)).

Host cells are transfected and preferably transformed with theabove-described expression or cloning vectors of this invention andcultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transfection refers to the taking up of an expression vector by a hostcell whether or not any coding sequences are in fact expressed. Numerousmethods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ precipitation and electroporation. Successfultransfection is generally recognized when any indication of theoperation of this vector occurs within the host cell. Methods fortransfection are well known in the art, and some are further describedherein.

Transformation means introducing DNA into an organism so that the DNA isreplicable, either as an extrachromosomal element or by chromosomalintegrant. Depending on the host cell used, transformation is done usingstandard techniques appropriate to such cells. Methods fortransformation are well known in the art, and some are further describedherein.

Prokaryotic host cells used to produce the FGFR3 can be cultured asdescribed generally in Sambrook et al., supra.

The mammalian host cells used to produce the FGFR3 can be cultured in avariety of media, which is well known in the art and some of which isdescribed herein.

The host cells referred to in this disclosure encompass cells in invitro culture as well as cells that are within a host animal.

Purification of FGFR3 may be accomplished using art-recognized methods,some of which are described herein.

The purified FGFR3 can be attached to a suitable matrix such as agarosebeads, acrylamide beads, glass beads, cellulose, various acryliccopolymers, hydroxyl methacrylate gels, polyacrylic and polymethacryliccopolymers, nylon, neutral and ionic carriers, and the like, for use inthe affinity chromatographic separation of phage display clones.Attachment of the FGFR3 protein to the matrix can be accomplished by themethods described in Methods in Enzymology, vol. 44 (1976). A commonlyemployed technique for attaching protein ligands to polysaccharidematrices, e.g. agarose, dextran or cellulose, involves activation of thecarrier with cyanogen halides and subsequent coupling of the peptideligand's primary aliphatic or aromatic amines to the activated matrix.

Alternatively, FGFR3 can be used to coat the wells of adsorption plates,expressed on host cells affixed to adsorption plates or used in cellsorting, or conjugated to biotin for capture with streptavidin-coatedbeads, or used in any other art-known method for panning phage displaylibraries.

The phage library samples are contacted with immobilized FGFR3 underconditions suitable for binding of at least a portion of the phageparticles with the adsorbent. Normally, the conditions, including pH,ionic strength, temperature and the like are selected to mimicphysiological conditions. The phages bound to the solid phase are washedand then eluted by acid, e.g. as described in Barbas et al., Proc. Natl.Acad. Sci USA, 88: 7978-7982 (1991), or by alkali, e.g. as described inMarks et al., J. Mol. Biol., 222: 581-597 (1991), or by FGFR3 antigencompetition, e.g. in a procedure similar to the antigen competitionmethod of Clackson et al., Nature, 352: 624-628 (1991). Phages can beenriched 20-1,000-fold in a single round of selection. Moreover, theenriched phages can be grown in bacterial culture and subjected tofurther rounds of selection.

The efficiency of selection depends on many factors, including thekinetics of dissociation during washing, and whether multiple antibodyfragments on a single phage can simultaneously engage with antigen.Antibodies with fast dissociation kinetics (and weak binding affinities)can be retained by use of short washes, multivalent phage display andhigh coating density of antigen in solid phase. The high density notonly stabilizes the phage through multivalent interactions, but favorsrebinding of phage that has dissociated. The selection of antibodieswith slow dissociation kinetics (and good binding affinities) can bepromoted by use of long washes and monovalent phage display as describedin Bass et al., Proteins, 8: 309-314 (1990) and in WO 92/09690, and alow coating density of antigen as described in Marks et al.,Biotechnol., 10: 779-783 (1992).

It is possible to select between phage antibodies of differentaffinities, even with affinities that differ slightly, for FGFR3.However, random mutation of a selected antibody (e.g. as performed insome of the affinity maturation techniques described above) is likely togive rise to many mutants, most binding to antigen, and a few withhigher affinity. With limiting FGFR3, rare high affinity phage could becompeted out. To retain all the higher affinity mutants, phages can beincubated with excess biotinylated FGFR3, but with the biotinylatedFGFR3 at a concentration of lower molarity than the target molaraffinity constant for FGFR3. The high affinity-binding phages can thenbe captured by streptavidin-coated paramagnetic beads. Such “equilibriumcapture” allows the antibodies to be selected according to theiraffinities of binding, with sensitivity that permits isolation of mutantclones with as little as two-fold higher affinity from a great excess ofphages with lower affinity. Conditions used in washing phages bound to asolid phase can also be manipulated to discriminate on the basis ofdissociation kinetics.

FGFR3 clones may be activity selected. In one embodiment, the inventionprovides FGFR3 antibodies that block the binding between a FGFR3receptor and its ligand (such as FGF1 and/or FGF9). Fv clonescorresponding to such FGFR3 antibodies can be selected by (1) isolatingFGFR3 clones from a phage library as described above, and optionallyamplifying the isolated population of phage clones by growing up thepopulation in a suitable bacterial host; (2) selecting FGFR3 and asecond protein against which blocking and non-blocking activity,respectively, is desired; (3) adsorbing the anti-FGFR3 phage clones toimmobilized FGFR3; (4) using an excess of the second protein to eluteany undesired clones that recognize FGFR3-binding determinants whichoverlap or are shared with the binding determinants of the secondprotein; and (5) eluting the clones which remain adsorbed following step(4). Optionally, clones with the desired blocking/non-blockingproperties can be further enriched by repeating the selection proceduresdescribed herein one or more times.

DNA encoding the hybridoma-derived monoclonal antibodies or phagedisplay Fv clones of the invention is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide primersdesigned to specifically amplify the heavy and light chain codingregions of interest from hybridoma or phage DNA template). Onceisolated, the DNA can be placed into expression vectors, which are thentransfected into host cells such as E. coli cells, simian COS cells,Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, to obtain the synthesis of thedesired monoclonal antibodies in the recombinant host cells. Reviewarticles on recombinant expression in bacteria of antibody-encoding DNAinclude Skerra et al., Curr. Opinion in Immunol., 5: 256 (1993) andPluckthun, Immunol. Revs, 130:151 (1992).

DNA encoding the Fv clones of the invention can be combined with knownDNA sequences encoding heavy chain and/or light chain constant regions(e.g., the appropriate DNA sequences can be obtained from Kabat et al.,supra) to form clones encoding full or partial length heavy and/or lightchains. It will be appreciated that constant regions of any isotype canbe used for this purpose, including IgG, IgM, IgA, IgD, and IgE constantregions, and that such constant regions can be obtained from any humanor animal species. A Fv clone derived from the variable domain DNA ofone animal (such as human) species and then fused to constant region DNAof another animal species to form coding sequence(s) for “hybrid,” fulllength heavy chain and/or light chain is included in the definitionof“chimeric” and “hybrid” antibody as used herein. In a preferredembodiment, a Fv clone derived from human variable DNA is fused to humanconstant region DNA to form coding sequence(s) for all human, full orpartial length heavy and/or light chains.

DNA encoding anti-FGFR3 antibody derived from a hybridoma of theinvention can also be modified, for example, by substituting the codingsequence for human heavy- and light-chain constant domains in place ofhomologous murine sequences derived from the hybridoma clone (e.g., asin the method of Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). DNA encoding a hybridoma or Fv clone-derivedantibody or fragment can be further modified by covalently joining tothe immunoglobulin coding sequence all or part of the coding sequencefor a non-immunoglobulin polypeptide. In this manner, “chimeric” or“hybrid” antibodies are prepared that have the binding specificity ofthe Fv clone or hybridoma clone-derived antibodies of the invention.

Antibody Fragments

The present invention encompasses antibody fragments. In certaincircumstances there are advantages of using antibody fragments, ratherthan whole antibodies. The smaller size of the fragments allows forrapid clearance, and may lead to improved access to solid tumors.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992); and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. Fab, Fv and ScFv antibodyfragments can all be expressed in and secreted from E. coli, thusallowing the facile production of large amounts of these fragments.Antibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)₂ fragments(Carter et al., Bio/Technology 10:163-167 (1992)). According to anotherapproach, F(ab′)₂ fragments can be isolated directly from recombinanthost cell culture. Fab and F(ab′)₂ fragment with increased in vivohalf-life comprising a salvage receptor binding epitope residues aredescribed in U.S. Pat. No. 5,869,046. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner. In other embodiments, the antibody of choice is a singlechain Fv fragment (scFv) (see, e.g., WO 93/16185; U.S. Pat. Nos.5,571,894 and 5,587,458). Fv and sFv are the only species with intactcombining sites that are devoid of constant regions; thus, they aresuitable for reduced nonspecific binding during in vivo use. sFv fusionproteins may be constructed to yield fusion of an effector protein ateither the amino or the carboxy terminus of an sFv. See AntibodyEngineering, ed. Borrebaeck, supra. The antibody fragment may also be a“linear antibody,” e.g., as described, for example, in U.S. Pat. No.5,641,870. Such linear antibody fragments may be monospecific orbispecific.

Humanized Antibodies

The present invention encompasses humanized antibodies. Various methodsfor humanizing non-human antibodies are known in the art. For example, ahumanized antibody can have one or more amino acid residues introducedinto it from a source which is non-human.

These non-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain.Humanization can be essentially performed following the method of Winterand co-workers (Jones et al. (1986) Nature 321:522-525; Riechmann et al.(1988) Nature 332:323-327; Verhoeyen et al. (1988) Science239:1534-1536), by substituting hypervariable region sequences for thecorresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567)wherein substantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome hypervariable region residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework for the humanized antibody (Sims et al. (1993) J.Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol. 196:901. Anothermethod uses a particular framework derived from the consensus sequenceof all human antibodies of a particular subgroup of light or heavychains. The same framework may be used for several different humanizedantibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285;Presta et al. (1993) J. Immunol., 151:2623.

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to one method, humanized antibodies areprepared by a process of analysis of the parental sequences and variousconceptual humanized products using three-dimensional models of theparental and humanized sequences. Three-dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart. Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e., the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen. In this way, FR residues can be selected and combined from therecipient and import sequences so that the desired antibodycharacteristic, such as increased affinity for the target antigen(s), isachieved. In general, the hypervariable region residues are directly andmost substantially involved in influencing antigen binding.

Human Antibodies

Human anti-FGFR3 antibodies of the invention can be constructed bycombining Fv clone variable domain sequence(s) selected fromhuman-derived phage display libraries with known human constant domainsequences(s) as described above. Alternatively, human monoclonalanti-FGFR3 antibodies of the invention can be made by the hybridomamethod. Human myeloma and mouse-human heteromyeloma cell lines for theproduction of human monoclonal antibodies have been described, forexample, by Kozbor J. Immunol., 133:3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63(Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol.,147:86 (1991).

It is now possible to produce transgenic animals (e.g., mice) that arecapable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy-chain joining region (JH) gene in chimeric and germ-linemutant mice results in complete inhibition of endogenous antibodyproduction. Transfer of the human germ-line immunoglobulin gene array insuch germ-line mutant mice will result in the production of humanantibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc.Natl. Acad. Sci USA, 90: 2551 (1993); Jakobovits et al., Nature, 362:255 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993).

Gene shuffling can also be used to derive human antibodies fromnon-human, e.g., rodent, antibodies, where the human antibody hassimilar affinities and specificities to the starting non-human antibody.According to this method, which is also called “epitope imprinting,”either the heavy or light chain variable region of a non-human antibodyfragment obtained by phage display techniques as described above isreplaced with a repertoire of human V domain genes, creating apopulation of non-human chain/human chain scFv or Fab chimeras.Selection with antigen results in isolation of a non-human chain/humanchain chimeric scFv or Fab wherein the human chain restores the antigenbinding site destroyed upon removal of the corresponding non-human chainin the primary phage display clone, i.e. the epitope governs (imprints)the choice of the human chain partner. When the process is repeated inorder to replace the remaining non-human chain, a human antibody isobtained (see PCT WO 93/06213 published Apr. 1, 1993). Unliketraditional humanization of non-human antibodies by CDR grafting, thistechnique provides completely human antibodies, which have no FR or CDRresidues of non-human origin.

Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forFGFR3 and the other is for any other antigen. Exemplary bispecificantibodies may bind to two different epitopes of the FGFR3. Bispecificantibodies may also be used to localize cytotoxic agents to cells whichexpress FGFR3. These antibodies possess an FGFR3-binding arm and an armwhich binds the cytotoxic agent (e.g., saporin, anti-interferon-α, vincaalkaloid, ricin A chain, methotrexate or radioactive isotope hapten).Bispecific antibodies can be prepared as full length antibodies orantibody fragments (e.g., F(ab′)₂ bispecific antibodies).

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305: 537 (1983)). Because of the random assortmentof immunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. The purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829 published May 13, 1993, and inTraunecker et al., EMBO J., 10: 3655 (1991).

According to a different and more preferred approach, antibody variabledomains with the desired binding specificities (antibody-antigencombining sites) are fused to immunoglobulin constant domain sequences.The fusion preferably is with an immunoglobulin heavy chain constantdomain, comprising at least part of the hinge, CH2, and CH3 regions. Itis preferred to have the first heavy-chain constant region (CH1),containing the site necessary for light chain binding, present in atleast one of the fusions. DNAs encoding the immunoglobulin heavy chainfusions and, if desired, the immunoglobulin light chain, are insertedinto separate expression vectors, and are co-transfected into a suitablehost organism. This provides for great flexibility in adjusting themutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986).

According to another approach, the interface between a pair of antibodymolecules can be engineered to maximize the percentage of heterodimerswhich are recovered from recombinant cell culture. The preferredinterface comprises at least a part of the CH3 domain of an antibodyconstant domain. In this method, one or more small amino acid sidechains from the interface of the first antibody molecule are replacedwith larger side chains (e.g., tyrosine or tryptophan). Compensatory“cavities” of identical or similar size to the large side chain(s) arecreated on the interface of the second antibody molecule by replacinglarge amino acid side chains with smaller ones (e.g., alanine orthreonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/00373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the HER2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (VH) connected to a light-chain variabledomain (VL) by a linker which is too short to allow pairing between thetwo domains on the same chain. Accordingly, the VH and VL domains of onefragment are forced to pair with the complementary VL and VH domains ofanother fragment, thereby forming two antigen-binding sites. Anotherstrategy for making bispecific antibody fragments by the use ofsingle-chain Fv (sFv) dimers has also been reported. See Gruber et al.,J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60(1991).

Multivalent Antibodies

A multivalent antibody may be internalized (and/or catabolized) fasterthan a bivalent antibody by a cell expressing an antigen to which theantibodies bind. The antibodies of the present invention can bemultivalent antibodies (which are other than of the IgM class) withthree or more antigen binding sites (e.g. tetravalent antibodies), whichcan be readily produced by recombinant expression of nucleic acidencoding the polypeptide chains of the antibody. The multivalentantibody can comprise a dimerization domain and three or more antigenbinding sites. The preferred dimerization domain comprises (or consistsof) an Fc region or a hinge region. In this scenario, the antibody willcomprise an Fc region and three or more antigen binding sitesamino-terminal to the Fe region. The preferred multivalent antibodyherein comprises (or consists of) three to about eight, but preferablyfour, antigen binding sites. The multivalent antibody comprises at leastone polypeptide chain (and preferably two polypeptide chains), whereinthe polypeptide chain(s) comprise two or more variable domains. Forinstance, the polypeptide chain(s) may comprise VD1-(X1)n-VD2-(X2)n-Fc,wherein VD1 is a first variable domain, VD2 is a second variable domain,Fc is one polypeptide chain of an Fc region, X1 and X2 represent anamino acid or polypeptide, and n is 0 or 1. For instance, thepolypeptide chain(s) may comprise: VH-CH1-flexible linker-VH-CH1-Fcregion chain; or VH-CH1-VH-CH1-Fc region chain. The multivalent antibodyherein preferably further comprises at least two (and preferably four)light chain variable domain polypeptides. The multivalent antibodyherein may, for instance, comprise from about two to about eight lightchain variable domain polypeptides. The light chain variable domainpolypeptides contemplated here comprise a light chain variable domainand, optionally, further comprise a CL domain.

Antibody Variants

In some embodiments, amino acid sequence modification(s) of theantibodies described herein are contemplated. For example, it may bedesirable to improve the binding affinity and/or other biologicalproperties of the antibody. Amino acid sequence variants of the antibodyare prepared by introducing appropriate nucleotide changes into theantibody nucleic acid, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of, residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution ismade to arrive at the final construct, provided that the final constructpossesses the desired characteristics. The amino acid alterations may beintroduced in the subject antibody amino acid sequence at the time thatsequence is made.

A useful method for identification of certain residues or regions of theantibody that are preferred locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells (1989)Science, 244:1081-1085. Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (mostpreferably alanine or polyalanine) to affect the interaction of theamino acids with antigen. Those amino acid locations demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other variants at, or for, the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressedimmunoglobulins are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody fusedto a cytotoxic polypeptide. Other insertional variants of the antibodymolecule include the fusion to the N- or C-terminus of the antibody toan enzyme (e.g., for ADEPT) or a polypeptide which increases the serumhalf-life of the antibody.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. For example, antibodies with a maturecarbohydrate structure that lacks fucose attached to an Fc region of theantibody are described in US Pat Appl No US 2003/0157108 (Presta, L.).See also US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Antibodies with abisecting N-acetylglucosamine (GlcNAc) in the carbohydrate attached toan Fc region of the antibody are referenced in WO 2003/011878,Jean-Mairet et al. and U.S. Pat. No. 6,602,684, Umana et al. Antibodieswith at least one galactose residue in the oligosaccharide attached toan Fc region of the antibody are reported in WO 1997/30087, Patel et al.See, also, WO 1998/58964 (Raju, S.) and WO 1999/22764 (Raju, S.)concerning antibodies with altered carbohydrate attached to the Fcregion thereof. See also US 2005/0123546 (Umana et al.) onantigen-binding molecules with modified glycosylation.

The preferred glycosylation variant herein comprises an Fc region,wherein a carbohydrate structure attached to the Fc region lacks fucose.Such variants have improved ADCC function. Optionally, the Fc regionfurther comprises one or more amino acid substitutions therein whichfurther improve ADCC, for example, substitutions at positions 298, 333,and/or 334 of the Fc region (Eu numbering of residues). Examples ofpublications related to “defucosylated” or “fucose-deficient” antibodiesinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; Okazaki et al. J. Mol. Biol.336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614(2004). Examples of cell lines producing defucosylated antibodiesinclude Lec13 CHO cells deficient in protein fucosylation (Ripka et al.Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al.,especially at Example 11), and knockout cell lines, such asalpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells(Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004)).

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid (at least two, at least three, atleast 4 or more) residue in the antibody molecule replaced by adifferent residue. The sites of greatest interest for substitutionalmutagenesis include the hypervariable regions, but FR alterations arealso contemplated. Conservative substitutions are shown in Table 1 underthe heading of “preferred substitutions.” If such substitutions resultin a change in biological activity, then more substantial changes,denominated “exemplary substitutions” in Table 1, or as furtherdescribed below in reference to amino acid classes, may be introducedand the products screened.

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Leu Phe; Norleucine Leu (L) Norleucine;Ile; Val; Ile Met; Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Leu Ala; Norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

-   -   (1) hydrophobic: norleucine, met, ala, val, leu, ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   (3) acidic: asp, glu;    -   (4) basic: his, lys, arg;    -   (5) residues that influence chain orientation: gly, pro; and    -   (6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g., a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther development will have improved biological properties relative tothe parent antibody from which they are generated. A convenient way forgenerating such substitutional variants involves affinity maturationusing phage display. Briefly, several hypervariable region sites (e.g.,6-7 sites) are mutated to generate all possible amino acid substitutionsat each site. The antibodies thus generated are displayed fromfilamentous phage particles as fusions to the gene III product of M13packaged within each particle. The phage-displayed variants are thenscreened for their biological activity (e.g., binding affinity) asherein disclosed. In order to identify candidate hypervariable regionsites for modification, alanine scanning mutagenesis can be performed toidentify hypervariable region residues contributing significantly toantigen binding. Alternatively, or additionally, it may be beneficial toanalyze a crystal structure of the antigen-antibody complex to identifycontact points between the antibody and antigen. Such contact residuesand neighboring residues are candidates for substitution according tothe techniques elaborated herein. Once such variants are generated, thepanel of variants is subjected to screening as described herein andantibodies with superior properties in one or more relevant assays maybe selected for further development.

Nucleic acid molecules encoding amino acid sequence variants of theantibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of the antibody.

It may be desirable to introduce one or more amino acid modifications inan Fc region of the immunoglobulin polypeptides of the invention,thereby generating a Fc region variant. The Fc region variant maycomprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 orIgG4 Fc region) comprising an amino acid modification (e.g., asubstitution) at one or more amino acid positions including that of ahinge cysteine.

In accordance with this description and the teachings of the art, it iscontemplated that in some embodiments, an antibody used in methods ofthe invention may comprise one or more alterations as compared to thewild type counterpart antibody, e.g., in the Fc region. These antibodieswould nonetheless retain substantially the same characteristics requiredfor therapeutic utility as compared to their wild type counterpart. Forexample, it is thought that certain alterations can be made in the Fcregion that would result in altered (i.e., either improved ordiminished) C1q binding and/or Complement Dependent Cytotoxicity (CDC),e.g., as described in WO99/51642. See also Duncan & Winter Nature322:738-40 (1988); U.S. Pat. Nos. 5,648,260; 5,624,821; and WO94/29351concerning other examples of Fc region variants. WO00/42072 (Presta) andWO 2004/056312 (Lowman) describe antibody variants with improved ordiminished binding to FcRs. The content of these patent publications arespecifically incorporated herein by reference. See, also, Shields et al.J Biol. Chem. 9(2): 6591-6604 (2001). Antibodies with increased halflives and improved binding to the neonatal Fc receptor (FcRn), which isresponsible for the transfer of maternal IgGs to the fetus (Guyer etal., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249(1994)), are described in US2005/0014934A1 (Hinton et al.). Theseantibodies comprise an Fc region with one or more substitutions thereinwhich improve binding of the Fc region to FcRn. Polypeptide variantswith altered Fc region amino acid sequences and increased or decreasedC1q binding capability are described in U.S. Pat. No. 6,194,551B1,WO99/51642. The contents of those patent publications are specificallyincorporated herein by reference. See, also, Idusogie et al., J Immunol.164: 4178-4184 (2000).

Antibody Derivatives

The antibodies of the present invention can be further modified tocontain additional nonproteinaceous moieties that are known in the artand readily available. Preferably, the moieties suitable forderivatization of the antibody are water soluble polymers. Non-limitingexamples of water soluble polymers include, but are not limited to,polyethylene glycol (PEG), copolymers of ethylene glycol/propyleneglycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), and dextran or poly(n-vinyl pyrrolidone)polyethyleneglycol, propropylene glycol homopolymers, prolypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde mayhave advantages in manufacturing due to its stability in water. Thepolymer may be of any molecular weight, and may be branched orunbranched. The number of polymers attached to the antibody may vary,and if more than one polymers are attached, they can be the same ordifferent molecules. In general, the number and/or type of polymers usedfor derivatization can be determined based on considerations including,but not limited to, the particular properties or functions of theantibody to be improved, whether the antibody derivative will be used ina therapy under defined conditions, etc.

Screening for Antibodies with Desired Properties

The antibodies of the present invention can be characterized for theirphysical/chemical properties and biological functions by various assaysknown in the art (some of which are disclosed herein). In someembodiments, antibodies are characterized for any one or more ofreduction or blocking of FGF (such as FGF 1 and/or FGF9) binding,reduction or blocking of FGFR3 activation, reduction or blocking ofFGFR3 downstream molecular signaling, disruption or blocking of FGFR3binding to a ligand (e.g., FGF1, FGF9), reduction or blocking of FGFR3dimerization, promotion of formation of monomeric FGFR3, binding tomonomeric FGFR3, and/or treatment and/or prevention of a tumor, cellproliferative disorder or a cancer; and/or treatment or prevention of adisorder associated with FGFR3 expression and/or activity (such asincreased FGFR3 expression and/or activity). In some embodiments, theantibodies are screened for increased FGFR3 activation, increased FGFR3downstream molecule signaling, apoptotic activity, FGFR3down-regulation, and effector function (e.g., ADCC activity).

The purified antibodies can be further characterized by a series ofassays including, but not limited to, N-terminal sequencing, amino acidanalysis, non-denaturing size exclusion high pressure liquidchromatography (HPLC), mass spectrometry, ion exchange chromatographyand papain digestion.

In certain embodiments of the invention, the antibodies produced hereinare analyzed for their biological activity. In some embodiments, theantibodies of the present invention are tested for their antigen bindingactivity. The antigen binding assays that are known in the art and canbe used herein include without limitation any direct or competitivebinding assays using techniques such as western blots,radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoprecipitation assays, fluorescent immunoassays, andprotein A immunoassays. Illustrative antigen binding and other assay areprovided below in the Examples section.

If an anti-FGFR3 antibody that inhibits cell growth is desired, thecandidate antibody can be tested in in vitro and/or in vivo assays thatmeasure inhibition of cell growth. If an anti-FGFR3 antibody that doesor does not promote apoptosis is desired, the candidate antibody can betested in assays that measure apoptosis. Methods for examining growthand/or proliferation of a cancer cell, or determining apoptosis of acancer cell are well known in the art and some are described andexemplified herein. Exemplary methods for determining cell growth and/orproliferation and/or apoptosis include, for example, BrdU incorporationassay, MTT, [3H]-thymidine incorporation (e.g., TopCount assay(PerkinElmer)), cell viability assays (e.g., CellTiter-Glo (Promega)),DNA fragmentation assays, caspase activation assays, tryptan blueexclusion, chromatin morphology assays and the like.

In one embodiment, the present invention contemplates an antibody thatpossesses effector functions. In certain embodiments, the Fc activitiesof the antibody are measured. In vitro and/or in vivo cytotoxicityassays can be conducted to confirm the reduction/depletion of CDC and/orADCC activities. For example, Fc receptor (FcR) binding assays can beconducted to ensure that the antibody lacks FcγR binding (hence likelylacking ADCC activity), but retains FcRn binding ability. The primarycells for mediating ADCC, NK cells, express Fc(RIII only, whereasmonocytes express Fc(RI, Fc(RII and Fc(RIII. FcR expression onhematopoietic cells is summarized in Table 3 on page 464 of Ravetch andKinet, Annu. Rev. Immunol 9:457-92 (1991). An example of an in vitroassay to assess ADCC activity of a molecule of interest is described inU.S. Pat. No. 5,500,362 or 5,821,337. An assay to detect ADCC activityis also exemplified herein. Useful effector cells for such assaysinclude peripheral blood mononuclear cells (PBMC) and Natural Killer(NK) cells. Alternatively, or additionally, ADCC activity of themolecule of interest may be assessed in vivo, e.g., in a animal modelsuch as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).C1q binding assays may also be carried out to confirm that the antibodyis unable to bind C1q and hence lacks CDC activity. To assess complementactivation, a CDC assay, e.g., as described in Gazzano-Santoro et al.,J. Immunol. Methods 202:163 (1996), may be performed. FcRn binding andin vivo clearance/half life determinations can also be performed usingmethods known in the art, e.g., those described in the Examples section.

If an anti-FGFR3 antibody that binds monomeric FGFR3 is desired, thecandidate antibody can be tested in assays (such as in vitro assays)that measure binding to monomeric FGFR3 and promotion of the formationof monomeric FGFR3. Such assays are known in the art and some assays aredescribed and exemplified herein.

If an anti-FGFR3 antibody that inhibits FGFR3 dimerization is desired,the candidate antibody can be tested in dimerization assays, e.g., asdescribed and exemplified herein.

In some embodiments, the FGFR3 agonist function of the candidateantibody is determined. Methods for assessing agonist function oractivity of FGFR3 antibodies are known in the art and some are alsodescribed and exemplified herein.

In some embodiments, ability of an FGFR3 antibody to promote FGFR3receptor down-regulation is determined, e.g., using methods describedand exemplified herein. In one embodiment, FGFR3 antibody is incubatedwith suitable test cells, e.g., bladder cancer cell lines (e.g., RT112),and after a suitable period of time, cell lysates are harvested andexamined for total FGFR3 levels. FACS analysis may also be used toexamine surface FGFR3 receptor levels following incubation withcandidate FGFR3 antibodies

Vectors, Host Cells, and Recombinant Methods

For recombinant production of an antibody of the invention, the nucleicacid encoding it is isolated and inserted into a replicable vector forfurther cloning (amplification of the DNA) or for expression. DNAencoding the antibody is readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody). Many vectors are available. The choice ofvector depends in part on the host cell to be used. Generally, preferredhost cells are of either prokaryotic or eukaryotic (generally mammalian)origin. It will be appreciated that constant regions of any isotype canbe used for this purpose, including IgG, IgM, IgA, IgD, and IgE constantregions, and that such constant regions can be obtained from any humanor animal species.

a. Generating Antibodies Using Prokaryotic Host Cells:

i. Vector Construction

Polynucleotide sequences encoding polypeptide components of the antibodyof the invention can be obtained using standard recombinant techniques.Desired polynucleotide sequences may be isolated and sequenced fromantibody producing cells such as hybridoma cells. Alternatively,polynucleotides can be synthesized using nucleotide synthesizer or PCRtechniques. Once obtained, sequences encoding the polypeptides areinserted into a recombinant vector capable of replicating and expressingheterologous polynucleotides in prokaryotic hosts. Many vectors that areavailable and known in the art can be used for the purpose of thepresent invention. Selection of an appropriate vector will depend mainlyon the size of the nucleic acids to be inserted into the vector and theparticular host cell to be transformed with the vector. Each vectorcontains various components, depending on its function (amplification orexpression of heterologous polynucleotide, or both) and itscompatibility with the particular host cell in which it resides. Thevector components generally include, but are not limited to: an originof replication, a selection marker gene, a promoter, a ribosome bindingsite (RBS), a signal sequence, the heterologous nucleic acid insert anda transcription termination sequence.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using pBR322, a plasmid derived from an E. colispecies. pBR322 contains genes encoding ampicillin (Amp) andtetracycline (Tet) resistance and thus provides easy means foridentifying transformed cells. pBR322, its derivatives, or othermicrobial plasmids or bacteriophage may also contain, or be modified tocontain, promoters which can be used by the microbial organism forexpression of endogenous proteins. Examples of pBR322 derivatives usedfor expression of particular antibodies are described in detail inCarter et al., U.S. Pat. No. 5,648,237.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example,bacteriophage such as λGEM™-11 may be utilized in making a recombinantvector which can be used to transform susceptible host cells such as E.coli LE392.

The expression vector of the invention may comprise two or morepromoter-cistron pairs, encoding each of the polypeptide components. Apromoter is an untranslated regulatory sequence located upstream (5′) toa cistron that modulates its expression. Prokaryotic promoters typicallyfall into two classes, inducible and constitutive. Inducible promoter isa promoter that initiates increased levels of transcription of thecistron under its control in response to changes in the culturecondition, e.g., the presence or absence of a nutrient or a change intemperature.

A large number of promoters recognized by a variety of potential hostcells are well known. The selected promoter can be operably linked tocistron DNA encoding the light or heavy chain by removing the promoterfrom the source DNA via restriction enzyme digestion and inserting theisolated promoter sequence into the vector of the invention. Both thenative promoter sequence and many heterologous promoters may be used todirect amplification and/or expression of the target genes. In someembodiments, heterologous promoters are utilized, as they generallypermit greater transcription and higher yields of expressed target geneas compared to the native target polypeptide promoter.

Promoters suitable for use with prokaryotic hosts include the PhoApromoter, the (3-galactamase and lactose promoter systems, a tryptophan(trp) promoter system and hybrid promoters such as the tac or the trcpromoter. However, other promoters that are functional in bacteria (suchas other known bacterial or phage promoters) are suitable as well. Theirnucleotide sequences have been published, thereby enabling a skilledworker operably to ligate them to cistrons encoding the target light andheavy chains (Siebenlist et al., (1980) Cell 20: 269) using linkers oradaptors to supply any required restriction sites.

In one aspect of the invention, each cistron within the recombinantvector comprises a secretion signal sequence component that directstranslocation of the expressed polypeptides across a membrane. Ingeneral, the signal sequence may be a component of the vector, or it maybe a part of the target polypeptide DNA that is inserted into thevector. The signal sequence selected for the purpose of this inventionshould be one that is recognized and processed (i.e., cleaved by asignal peptidase) by the host cell. For prokaryotic host cells that donot recognize and process the signal sequences native to theheterologous polypeptides, the signal sequence is substituted by aprokaryotic signal sequence selected, for example, from the groupconsisting of the alkaline phosphatase, penicillinase, Ipp, orheat-stable enterotoxin II (STII) leaders, LamB, PhoE, PelB, OmpA, andMBP. In one embodiment of the invention, the signal sequences used inboth cistrons of the expression system are STII signal sequences orvariants thereof.

In another aspect, the production of the immunoglobulins according tothe invention can occur in the cytoplasm of the host cell, and thereforedoes not require the presence of secretion signal sequences within eachcistron. In that regard, immunoglobulin light and heavy chains areexpressed, folded and assembled to form functional immunoglobulinswithin the cytoplasm. Certain host strains (e.g., the E. colitrxB-strains) provide cytoplasm conditions that are favorable fordisulfide bond formation, thereby permitting proper folding and assemblyof expressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

Prokaryotic host cells suitable for expressing antibodies of theinvention include Archaebacteria and Eubacteria, such as Gram-negativeor Gram-positive organisms. Examples of useful bacteria includeEscherichia (e.g., E. coli), Bacilli (e.g., B. subtilis),Enterobacteria, Pseudomonas species (e.g., P. aeruginosa), Salmonellatyphimurium, Serratia marcescans, Klebsiella, Proteus, Shigella,Rhizobia, Vitreoscilla, or Paracoccus. In one embodiment, gram-negativecells are used. In one embodiment, E. coli cells are used as hosts forthe invention. Examples of E. coli strains include strain W3110(Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, D.C.:American Society for Microbiology, 1987), pp. 1190-1219; ATCC DepositNo. 27,325) and derivatives thereof, including strain 33D3 havinggenotype W3110 ΔfhuA (ΔtonA) ptr3 lac Iq lacL8 ΔompTA(nmpc-fepE) degP41kanR (U.S. Pat. No. 5,639,635). Other strains and derivatives thereof,such as E. coli 294 (ATCC 31,446), E. coli B, E. coli λ 1776 (ATCC31,537) and E. coli RV308(ATCC 31,608) are also suitable. These examplesare illustrative rather than limiting. Methods for constructingderivatives of any of the above-mentioned bacteria having definedgenotypes are known in the art and described in, for example, Bass etal., Proteins, 8:309-314 (1990). It is generally necessary to select theappropriate bacteria taking into consideration replicability of thereplicon in the cells of a bacterium. For example, E. coli, Serratia, orSalmonella species can be suitably used as the host when well knownplasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supplythe replicon. Typically the host cell should secrete minimal amounts ofproteolytic enzymes, and additional protease inhibitors may desirably beincorporated in the cell culture.

ii. Antibody Production

Host cells are transformed with the above-described expression vectorsand cultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transformation means introducing DNA into the prokaryotic host so thatthe DNA is replicable, either as an extrachromosomal element or bychromosomal integrant. Depending on the host cell used, transformationis done using standard techniques appropriate to such cells. The calciumtreatment employing calcium chloride is generally used for bacterialcells that contain substantial cell-wall barriers. Another method fortransformation employs polyethylene glycol/DMSO. Yet another techniqueused is electroporation.

Prokaryotic cells used to produce the polypeptides of the invention aregrown in media known in the art and suitable for culture of the selectedhost cells. Examples of suitable media include Luria broth (LB) plusnecessary nutrient supplements. In some embodiments, the media alsocontains a selection agent, chosen based on the construction of theexpression vector, to selectively permit growth of prokaryotic cellscontaining the expression vector. For example, ampicillin is added tomedia for growth of cells expressing ampicillin resistant gene.

Any necessary supplements besides carbon, nitrogen, and inorganicphosphate sources may also be included at appropriate concentrationsintroduced alone or as a mixture with another supplement or medium suchas a complex nitrogen source. Optionally the culture medium may containone or more reducing agents selected from the group consisting ofglutathione, cysteine, cystamine, thioglycollate, dithioerythritol anddithiothreitol.

The prokaryotic host cells are cultured at suitable temperatures. For E.coli growth, for example, the preferred temperature ranges from about20° C. to about 39° C., more preferably from about 25° C. to about 37°C., even more preferably at about 30° C. The pH of the medium may be anypH ranging from about 5 to about 9, depending mainly on the hostorganism. For E. coli, the pH is preferably from about 6.8 to about 7.4,and more preferably about 7.0.

If an inducible promoter is used in the expression vector of theinvention, protein expression is induced under conditions suitable forthe activation of the promoter. In one aspect of the invention, PhoApromoters are used for controlling transcription of the polypeptides.Accordingly, the transformed host cells are cultured in aphosphate-limiting medium for induction. Preferably, thephosphate-limiting medium is the C.R.A.P medium (see, e.g., Simmons etal., J. Immunol. Methods (2002), 263:133-147). A variety of otherinducers may be used, according to the vector construct employed, as isknown in the art.

In one embodiment, the expressed polypeptides of the present inventionare secreted into and recovered from the periplasm of the host cells.Protein recovery typically involves disrupting the microorganism,generally by such means as osmotic shock, sonication or lysis. Oncecells are disrupted, cell debris or whole cells may be removed bycentrifugation or filtration. The proteins may be further purified, forexample, by affinity resin chromatography. Alternatively, proteins canbe transported into the culture media and isolated therein. Cells may beremoved from the culture and the culture supernatant being filtered andconcentrated for further purification of the proteins produced. Theexpressed polypeptides can be further isolated and identified usingcommonly known methods such as polyacrylamide gel electrophoresis (PAGE)and Western blot assay.

In one aspect of the invention, antibody production is conducted inlarge quantity by a fermentation process. Various large-scale fed-batchfermentation procedures are available for production of recombinantproteins. Large-scale fermentations have at least 1000 liters ofcapacity, preferably about 1,000 to 100,000 liters of capacity. Thesefermentors use agitator impellers to distribute oxygen and nutrients,especially glucose (the preferred carbon/energy source). Small scalefermentation refers generally to fermentation in a fermentor that is nomore than approximately 100 liters in volumetric capacity, and can rangefrom about 1 liter to about 100 liters.

In a fermentation process, induction of protein expression is typicallyinitiated after the cells have been grown under suitable conditions to adesired density, e.g., an OD550 of about 180-220, at which stage thecells are in the early stationary phase. A variety of inducers may beused, according to the vector construct employed, as is known in the artand described above. Cells may be grown for shorter periods prior toinduction. Cells are usually induced for about 12-50 hours, althoughlonger or shorter induction time may be used.

To improve the production yield and quality of the polypeptides of theinvention, various fermentation conditions can be modified. For example,to improve the proper assembly and folding of the secreted antibodypolypeptides, additional vectors overexpressing chaperone proteins, suchas Dsb proteins (DsbA, DsbB, DsbC, DsbD, and/or DsbG) or FkpA (apeptidylprolyl cis,trans-isomerase with chaperone activity) can be usedto co-transform the host prokaryotic cells. The chaperone proteins havebeen demonstrated to facilitate the proper folding and solubility ofheterologous proteins produced in bacterial host cells. Chen et al.,(1999) J. Biol. Chem. 274:19601-19605; Georgiou et al., U.S. Pat. No.6,083,715; Georgiou et al., U.S. Pat. No. 6,027,888; Bothmann andPluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun,(2000) J. Biol. Chem. 275:17106-17113; Arie et al., (2001) Mol.Microbiol. 39:199-210.

To minimize proteolysis of expressed heterologous proteins (especiallythose that are proteolytically sensitive), certain host strainsdeficient for proteolytic enzymes can be used for the present invention.For example, host cell strains may be modified to effect geneticmutation(s) in the genes encoding known bacterial proteases such asProtease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V,Protease VI, and combinations thereof. Some E. coli protease-deficientstrains are available and described in, for example, Joly et al.,(1998), supra; Georgiou et al., U.S. Pat. No. 5,264,365; Georgiou etal., U.S. Pat. No. 5,508,192; Hara et al., Microbial Drug Resistance,2:63-72 (1996).

In one embodiment, E. coli strains deficient for proteolytic enzymes andtransformed with plasmids overexpressing one or more chaperone proteinsare used as host cells in the expression system of the invention.

iii. Antibody Purification

Standard protein purification methods known in the art can be employed.The following procedures are exemplary of suitable purificationprocedures: fractionation on immunoaffinity or ion-exchange columns,ethanol precipitation, reverse phase HPLC, chromatography on silica oron a cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE,ammonium sulfate precipitation, and gel filtration using, for example,Sephadex G-75.

In one aspect, Protein A immobilized on a solid phase is used forimmunoaffinity purification of the full length antibody products of theinvention. Protein A is a 41 kD cell wall protein from Staphylococcusaureas which binds with a high affinity to the Fc region of antibodies.Lindmark et al., (1983) J. Immunol. Meth. 62:1-13. The solid phase towhich Protein A is immobilized is preferably a column comprising a glassor silica surface, more preferably a controlled pore glass column or asilicic acid column. In some applications, the column has been coatedwith a reagent, such as glycerol, in an attempt to prevent nonspecificadherence of contaminants.

As the first step of purification, the preparation derived from the cellculture as described above is applied onto the Protein A immobilizedsolid phase to allow specific binding of the antibody of interest toProtein A. The solid phase is then washed to remove contaminantsnon-specifically bound to the solid phase. Finally the antibody ofinterest is recovered from the solid phase by elution.

b. Generating Antibodies Using Eukaryotic Host Cells:

The vector components generally include, but are not limited to, one ormore of the following: a signal sequence, an origin of replication, oneor more marker genes, an enhancer element, a promoter, and atranscription termination sequence.

(i) Signal Sequence Component

A vector for use in a eukaryotic host cell may also contain a signalsequence or other polypeptide having a specific cleavage site at theN-terminus of the mature protein or polypeptide of interest. Theheterologous signal sequence selected preferably is one that isrecognized and processed (i.e., cleaved by a signal peptidase) by thehost cell. In mammalian cell expression, mammalian signal sequences aswell as viral secretory leaders, for example, the herpes simplex gDsignal, are available.

The DNA for such precursor region is ligated in reading frame to DNAencoding the antibody.

(ii) Origin of Replication

Generally, an origin of replication component is not needed formammalian expression vectors. For example, the SV40 origin may typicallybe used only because it contains the early promoter.

(iii) Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, where relevant, or (c) supply critical nutrients notavailable from complex media.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theantibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-Iand -II, preferably primate metallothionein genes, adenosine deaminase,ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity (e.g., ATCCCRL-9096).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding an antibody, wild-type DHFR protein, and another selectablemarker such as aminoglycoside 3′-phosphotransferase (APH) can beselected by cell growth in medium containing a selection agent for theselectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

(iv) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the antibodypolypeptide nucleic acid. Promoter sequences are known for eukaryotes.Virtually alleukaryotic genes have an AT-rich region locatedapproximately 25 to 30 bases upstream from the site where transcriptionis initiated. Another sequence found 70 to 80 bases upstream from thestart of transcription of many genes is a CNCAAT region where N may beany nucleotide. At the 3′ end of most eukaryotic genes is an AATAAAsequence that may be the signal for addition of the poly A tail to the3′ end of the coding sequence. All of these sequences are suitablyinserted into eukaryotic expression vectors.

Antibody polypeptide transcription from vectors in mammalian host cellsis controlled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus, and Simian Virus 40(SV40), from heterologous mammalian promoters, e.g., the actin promoteror an immunoglobulin promoter, from heat-shock promoters, provided suchpromoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. Alternatively, the Rous Sarcoma Virus long terminal repeatcan be used as the promoter.

(v) Enhancer Element Component

Transcription of DNA encoding the antibody polypeptide of this inventionby higher eukaryotes is often increased by inserting an enhancersequence into the vector. Many enhancer sequences are now known frommammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin).Typically, however, one will use an enhancer from a eukaryotic cellvirus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theantibody polypeptide-encoding sequence, but is preferably located at asite 5′ from the promoter.

(vi) Transcription Termination Component

Expression vectors used in eukaryotic host cells will typically alsocontain sequences necessary for the termination of transcription and forstabilizing the mRNA. Such sequences are commonly available from the 5′and, occasionally 3′, untranslated regions of eukaryotic or viral DNAsor cDNAs. These regions contain nucleotide segments transcribed aspolyadenylated fragments in the untranslated portion of the mRNAencoding an antibody. One useful transcription termination component isthe bovine growth hormone polyadenylation region. See WO94/11026 and theexpression vector disclosed therein.

(vii) Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein include higher eukaryote cells described herein, includingvertebrate host cells. Propagation of vertebrate cells in culture(tissue culture) has become a routine procedure. Examples of usefulmammalian host cell lines are monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture, Graham et al., J. Gen.Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10);Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad.Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol.Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70);African green monkey kidney cells (VERO-76, ATCC CRL-1587); humancervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK,ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); humanlung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065);mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al.,Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and ahuman hepatoma line (Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

(viii) Culturing the Host Cells

The host cells used to produce an antibody of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othernecessary supplements may also be included at appropriate concentrationsthat would be known to those skilled in the art. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan.

(ix) Purification of Antibody

When using recombinant techniques, the antibody can be producedintracellularly, or directly secreted into the medium. If the antibodyis produced intracellularly, as a first step, the particulate debris,either host cells or lysed fragments, are removed, for example, bycentrifugation or ultrafiltration. Where the antibody is secreted intothe medium, supernatants from such expression systems are generallyfirst concentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. A protease inhibitor such as PMSF may be included in any of theforegoing steps to inhibit proteolysis and antibiotics may be includedto prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a CH3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

Immunoconjugates

The invention also provides immunoconjugates (interchangeably termed“antibody-drug conjugates” or “ADC”), comprising any of the anti-FGFR3antibodies described herein conjugated to a cytotoxic agent such as achemotherapeutic agent, a drug, a growth inhibitory agent, a toxin(e.g., an enzymatically active toxin of bacterial, fungal, plant, oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate).

The use of antibody-drug conjugates for the local delivery of cytotoxicor cytostatic agents, i.e., drugs to kill or inhibit tumor cells in thetreatment of cancer (Syrigos and Epenetos (1999) Anticancer Research19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drg. Del. Rev.26:151-172; U.S. Pat. No. 4,975,278) allows targeted delivery of thedrug moiety to tumors, and intracellular accumulation therein, wheresystemic administration of these unconjugated drug agents may result inunacceptable levels of toxicity to normal cells as well as the tumorcells sought to be eliminated (Baldwin et al., (1986) Lancet pp. (Mar.15, 1986):603-05; Thorpe, (1985) “Antibody Carriers Of Cytotoxic AgentsIn Cancer Therapy: A Review,” in Monoclonal Antibodies '84: BiologicalAnd Clinical Applications, A. Pinchera et al. (ed.s), pp. 475-506).Maximal efficacy with minimal toxicity is sought thereby. Bothpolyclonal antibodies and monoclonal antibodies have been reported asuseful in these strategies (Rowland et al., (1986) Cancer Immunol.Immunother., 21:183-87). Drugs used in these methods include daunomycin,doxorubicin, methotrexate, and vindesine (Rowland et al., (1986) supra).Toxins used in antibody-toxin conjugates include bacterial toxins suchas diphtheria toxin, plant toxins such as ricin, small molecule toxinssuch as geldanamycin (Mandler et al (2000) Jour. of the Nat. CancerInst. 92(19):1573-1581; Mandler et al., (2000) Bioorganic & Med. Chem.Letters 10:1025-1028; Mandler et al., (2002) Bioconjugate Chem.13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl.Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al., (1998)Cancer Res. 58:2928; Hinman et al., (1993) Cancer Res. 53:3336-3342).The toxins may effect their cytotoxic and cytostatic effects bymechanisms including tubulin binding, DNA binding, or topoisomeraseinhibition. Some cytotoxic drugs tend to be inactive or less active whenconjugated to large antibodies or protein receptor ligands.

ZEVALIN® (ibritumomab tiuxetan, Biogen/Idec) is an antibody-radioisotopeconjugate composed of a murine IgG1 kappa monoclonal antibody directedagainst the CD20 antigen found on the surface of normal and malignant Blymphocytes and ¹¹¹In or ⁹⁰Y radioisotope bound by a thiourealinker-chelator (Wiseman et al., (2000) Eur. Jour. Nucl. Med.27(7):766-77; Wiseman et al., (2002) Blood 99(12):4336-42; Witzig etal., (2002) J. Clin. Oncol. 20(10):2453-63; Witzig et al., (2002) J.Clin. Oncol. 20(15):3262-69). Although ZEVALIN has activity againstB-cell non-Hodgkin's Lymphoma (NHL), administration results in severeand prolonged cytopenias in most patients. MYLOTARG™ (gemtuzumabozogamicin, Wyeth Pharmaceuticals), an antibody drug conjugate composedof a hu CD33 antibody linked to calicheamicin, was approved in 2000 forthe treatment of acute myeloid leukemia by injection (Drugs of theFuture (2000) 25(7):686; U.S. Pat. Nos. 4,970,198; 5,079,233; 5,585,089;5,606,040; 5,6937,62; 5,739,116; 5,767,285; 5,773,001). Cantuzumabmertansine (Immunogen, Inc.), an antibody drug conjugate composed of thehuC242 antibody linked via the disulfide linker SPP to the maytansinoiddrug moiety, DM1, is advancing into Phase II trials for the treatment ofcancers that express CanAg, such as colon, pancreatic, gastric, andothers. MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.), anantibody drug conjugate composed of the anti-prostate specific membraneantigen (PSMA) monoclonal antibody linked to the maytansinoid drugmoiety, DM1, is under development for the potential treatment ofprostate tumors. The auristatin peptides, auristatin E (AE) andmonomethylauristatin (MMAE), synthetic analogs of dolastatin, wereconjugated to chimeric monoclonal antibodies cBR96 (specific to Lewis Yon carcinomas) and cAC10 (specific to CD30 on hematologicalmalignancies) (Doronina et al., (2003) Nature Biotechnology21(7):778-784) and are under therapeutic development.

Chemotherapeutic agents useful in the generation of immunoconjugates aredescribed herein (e.g., above). Enzymatically active toxins andfragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), Momordica charantiainhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.See, e.g., WO 93/21232 published Oct. 28, 1993. A variety ofradionuclides are available for the production of radioconjugatedantibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl suberate),aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

Conjugates of an antibody and one or more small molecule toxins, such asa calicheamicin, maytansinoids, dolastatins, aurostatins, atrichothecene, and CC1065, and the derivatives of these toxins that havetoxin activity, are also contemplated herein.

i. Maytansine and Maytansinoids

In some embodiments, the immunoconjugate comprises an antibody (fulllength or fragments) of the invention conjugated to one or moremaytansinoid molecules.

Maytansinoids are mitototic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinol and derivatives and analogues thereof aredisclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870;4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268;4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348;4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and4,371,533.

Maytansinoid drug moieties are attractive drug moieties in antibody drugconjugates because they are: (i) relatively accessible to prepare byfermentation or chemical modification, derivatization of fermentationproducts, (ii) amenable to derivatization with functional groupssuitable for conjugation through the non-disulfide linkers toantibodies, (iii) stable in plasma, and (iv) effective against a varietyof tumor cell lines.

Immunoconjugates containing maytansinoids, methods of making same, andtheir therapeutic use are disclosed, for example, in U.S. Pat. Nos.5,208,020, 5,416,064 and European Patent EP 0 425 235 B1, thedisclosures of which are hereby expressly incorporated by reference. Liuet al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) describedimmunoconjugates comprising a maytansinoid designated DM1 linked to themonoclonal antibody C242 directed against human colorectal cancer. Theconjugate was found to be highly cytotoxic towards cultured colon cancercells, and showed antitumor activity in an in vivo tumor growth assay.Chari et al., Cancer Research 52:127-131 (1992) describeimmunoconjugates in which a maytansinoid was conjugated via a disulfidelinker to the murine antibody A7 binding to an antigen on human coloncancer cell lines, or to another murine monoclonal antibody TA. 1 thatbinds the HER-2/neu oncogene. The cytotoxicity of the TA. 1-maytansinoidconjugate was tested in vitro on the human breast cancer cell lineSK-BR-3, which expresses 3×10⁵ HER-2 surface antigens per cell. The drugconjugate achieved a degree of cytotoxicity similar to the freemaytansinoid drug, which could be increased by increasing the number ofmaytansinoid molecules per antibody molecule. The A7-maytansinoidconjugate showed low systemic cytotoxicity in mice.

Antibody-maytansinoid conjugates are prepared by chemically linking anantibody to a maytansinoid molecule without significantly diminishingthe biological activity of either the antibody or the maytansinoidmolecule. See, e.g., U.S. Pat. No. 5,208,020 (the disclosure of which ishereby expressly incorporated by reference). An average of 3-4maytansinoid molecules conjugated per antibody molecule has shownefficacy in enhancing cytotoxicity of target cells without negativelyaffecting the function or solubility of the antibody, although even onemolecule of toxin/antibody would be expected to enhance cytotoxicityover the use of naked antibody. Maytansinoids are well known in the artand can be synthesized by known techniques or isolated from naturalsources. Suitable maytansinoids are disclosed, for example, in U.S. Pat.No. 5,208,020 and in the other patents and nonpatent publicationsreferred to hereinabove. Preferred maytansinoids are maytansinol andmaytansinol analogues modified in the aromatic ring or at otherpositions of the maytansinol molecule, such as various maytansinolesters.

There are many linking groups known in the art for makingantibody-maytansinoid conjugates, including, for example, thosedisclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, Chari etal., Cancer Research 52:127-131 (1992), and U.S. patent application Ser.No. 10/960,602, filed Oct. 8, 2004, the disclosures of which are herebyexpressly incorporated by reference. Antibody-maytansinoid conjugatescomprising the linker component SMCC may be prepared as disclosed inU.S. patent application Ser. No. 10/960,602, filed Oct. 8, 2004. Thelinking groups include disulfide groups, thioether groups, acid labilegroups, photolabile groups, peptidase labile groups, or esterase labilegroups, as disclosed in the above-identified patents, disulfide andthioether groups being preferred. Additional linking groups aredescribed and exemplified herein.

Conjugates of the antibody and maytansinoid may be made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agentsinclude N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlssonet al., Biochem. J. 173:723-737 (1978)) andN-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for adisulfide linkage.

The linker may be attached to the maytansinoid molecule at variouspositions, depending on the type of the link. For example, an esterlinkage may be formed by reaction with a hydroxyl group usingconventional coupling techniques. The reaction may occur at the C-3position having a hydroxyl group, the C-14 position modified withhydroxymethyl, the C-15 position modified with a hydroxyl group, and theC-20 position having a hydroxyl group. In a preferred embodiment, thelinkage is formed at the C-3 position of maytansinol or a maytansinolanalogue.

ii. Auristatins and Dolastatins

In some embodiments, the immunoconjugate comprises an antibody of theinvention conjugated to dolastatins or dolostatin peptidic analogs andderivatives, the auristatins (U.S. Pat. Nos. 5,635,483 and 5,780,588).Dolastatins and auristatins have been shown to interfere withmicrotubule dynamics, GTP hydrolysis, and nuclear and cellular division(Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584)and have anticancer (U.S. Pat. No. 5,663,149) and antifungal activity(Pettit et al., (1998) Antimicrob. Agents Chemother. 42:2961-2965). Thedolastatin or auristatin drug moiety may be attached to the antibodythrough the N (amino) terminus or the C (carboxyl) terminus of thepeptidic drug moiety (WO 02/088172).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties DE and DF, disclosed in“Monomethylvaline Compounds Capable of Conjugation to Ligands,” U.S.Ser. No. 10/983,340, filed Nov. 5, 2004, the disclosure of which isexpressly incorporated by reference in its entirety.

Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to the liquidphase synthesis method (see E. Schroder and K. Lübke, “The Peptides,”volume 1, pp. 76-136, 1965, Academic Press) that is well known in thefield of peptide chemistry. The auristatin/dolastatin drug moieties maybe prepared according to the methods of: U.S. Pat. Nos. 5,635,483 and5,780,588; Pettit et al., (1989) J. Am. Chem. Soc. 111:5463-5465; Pettitet al., (1998) Anti-Cancer Drug Design 13:243-277; Pettit, G. R., etal., Synthesis, 1996, 719-725; and Pettit et al., (1996) J. Chem. Soc.Perkin Trans. 1 5:859-863. See also Doronina (2003) Nat. Biotechnol.21(7):778-784; “Monomethylvaline Compounds Capable of Conjugation toLigands,” U.S. Ser. No. 10/983,340, filed Nov. 5, 2004, herebyincorporated by reference in its entirety (disclosing, e.g., linkers andmethods of preparing monomethylvaline compounds such as MMAE and MMAFconjugated to linkers).

iii. Calicheamicin

In other embodiments, the immunoconjugate comprises an antibody of theinvention conjugated to one or more calicheamicin molecules. Thecalicheamicin family of antibiotics are capable of producingdouble-stranded DNA breaks at sub-picomolar concentrations. For thepreparation of conjugates of the calicheamicin family, see U.S. Pat.Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710,5,773,001, and 5,877,296 (all to American Cyanamid Company). Structuralanalogues of calicheamicin which may be used include, but are notlimited to, γ₁ ^(I), α₂ ^(I), α₃ ^(I), N-acetyl-γ₁ ^(I), PSAG and θ^(I)₁ (Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al.,Cancer Research 58:2925-2928 (1998) and the aforementioned U.S. patentsto American Cyanamid). Another anti-tumor drug that the antibody can beconjugated is QFA which is an antifolate. Both calicheamicin and QFAhave intracellular sites of action and do not readily cross the plasmamembrane. Therefore, cellular uptake of these agents through antibodymediated internalization greatly enhances their cytotoxic effects.

iv. Other Cytotoxic Agents

Other antitumor agents that can be conjugated to the antibodies of theinvention include BCNU, streptozoicin, vincristine and 5-fluorouracil,the family of agents known collectively LL-E33288 complex described inU.S. Pat. Nos. 5,053,394 and 5,770,710, as well as esperamicins (U.S.Pat. No. 5,877,296).

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates an immunoconjugate formedbetween an antibody and a compound with nucleolytic activity (e.g., aribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated antibodies. Examples includeAt²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² andradioactive isotopes of Lu. When the conjugate is used for detection, itmay comprise a radioactive atom for scintigraphic studies, for exampletc^(99m) or I¹²³, or a spin label for nuclear magnetic resonance (NMR)imaging (also known as magnetic resonance imaging, mri), such asiodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13,nitrogen-15, oxygen-17, gadolinium, manganese or iron.

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as tc^(99m) or I¹²³, Re¹⁸⁶, Re¹⁸⁸ and In¹¹¹ can be attachedvia a cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al (1978) Biochem.Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

Conjugates of the antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of the cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Research 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The compounds of the invention expressly contemplate, but are notlimited to, ADC prepared with cross-linker reagents: BMPS, EMCS, GMBS,HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS,sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, andsulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which arecommercially available (e.g., from Pierce Biotechnology, Inc., Rockford,Ill., U.S.A). See pages 467-498, 2003-2004 Applications Handbook andCatalog.

v. Preparation of Antibody Drug Conjugates

In the antibody drug conjugates (ADC) of the invention, an antibody (Ab)is conjugated to one or more drug moieties (D), e.g. about 1 to about 20drug moieties per antibody, through a linker (L). The ADC of Formula Imay be prepared by several routes, employing organic chemistryreactions, conditions, and reagents known to those skilled in the art,including: (1) reaction of a nucleophilic group of an antibody with abivalent linker reagent, to form Ab-L, via a covalent bond, followed byreaction with a drug moiety D; and (2) reaction of a nucleophilic groupof a drug moiety with a bivalent linker reagent, to form D-L, via acovalent bond, followed by reaction with the nucleophilic group of anantibody. Additional methods for preparing ADC are described herein.

Ab-(L-D)_(p)  I

The linker may be composed of one or more linker components. Exemplarylinker components include 6-maleimidocaproyl (“MC”), maleimidopropanoyl(“MP”), valine-citrulline (“val-cit”), alanine-phenylalanine(“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), N-Succinimidyl4-(2-pyridylthio) pentanoate (“SPP”), N-Succinimidyl4-(N-maleimidomethyl) cyclohexane-1 carboxylate (“SMCC’), andN-Succinimidyl (4-iodo-acetyl) aminobenzoate (“SIAB”). Additional linkercomponents are known in the art and some are described herein. See also“Monomethylvaline Compounds Capable of Conjugation to Ligands,” U.S.Ser. No. 10/983,340, filed Nov. 5, 2004, the contents of which arehereby incorporated by reference in its entirety.

In some embodiments, the linker may comprise amino acid residues.Exemplary amino acid linker components include a dipeptide, atripeptide, a tetrapeptide or a pentapeptide. Exemplary dipeptidesinclude: valine-citrulline (vc or val-cit), alanine-phenylalanine (af orala-phe). Exemplary tripeptides include: glycine-valine-citrulline(gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acidresidues which comprise an amino acid linker component include thoseoccurring naturally, as well as minor amino acids and non-naturallyoccurring amino acid analogs, such as citrulline. Amino acid linkercomponents can be designed and optimized in their selectivity forenzymatic cleavage by a particular enzymes, for example, atumor-associated protease, cathepsin B, C and D, or a plasmin protease.

Nucleophilic groups on antibodies include, but are not limited to: (i)N-terminal amine groups, (ii) side chain amine groups, e.g. lysine,(iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl oramino groups where the antibody is glycosylated. Amine, thiol, andhydroxyl groups are nucleophilic and capable of reacting to formcovalent bonds with electrophilic groups on linker moieties and linkerreagents including: (i) active esters such as NHS esters, HOBt esters,haloformates, and acid halides; (ii) alkyl and benzyl halides such ashaloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimidegroups. Certain antibodies have reducible interchain disulfides, i.e.cysteine bridges. Antibodies may be made reactive for conjugation withlinker reagents by treatment with a reducing agent such as DTT(dithiothreitol). Each cysteine bridge will thus form, theoretically,two reactive thiol nucleophiles. Additional nucleophilic groups can beintroduced into antibodies through the reaction of lysines with2-iminothiolane (Traut's reagent) resulting in conversion of an amineinto a thiol. Reactive thiol groups may be introduced into the antibodyby introducing one, two, three, four, or more cysteine residues (e.g.,preparing mutant antibodies comprising one or more non-native cysteineamino acid residues).

Antibody drug conjugates of the invention may also be produced bymodification of the antibody to introduce electrophilic moieties, whichcan react with nucleophilic substituents on the linker reagent or drug.The sugars of glycosylated antibodies may be oxidized, e.g., withperiodate oxidizing reagents, to form aldehyde or ketone groups whichmay react with the amine group of linker reagents or drug moieties. Theresulting imine Schiff base groups may form a stable linkage, or may bereduced, e.g., by borohydride reagents to form stable amine linkages. Inone embodiment, reaction of the carbohydrate portion of a glycosylatedantibody with either galactose oxidase or sodium meta-periodate mayyield carbonyl (aldehyde and ketone) groups in the protein that canreact with appropriate groups on the drug (Hermanson, BioconjugateTechniques). In another embodiment, proteins containing N-terminalserine or threonine residues can react with sodium meta-periodate,resulting in production of an aldehyde in place of the first amino acid(Geoghegan & Stroh, (1992) Bioconjugate Chem. 3:138-146; U.S. Pat. No.5,362,852). Such aldehyde can be reacted with a drug moiety or linkernucleophile.

Likewise, nucleophilic groups on a drug moiety include, but are notlimited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groupscapable of reacting to form covalent bonds with electrophilic groups onlinker moieties and linker reagents including: (i) active esters such asNHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl andbenzyl halides such as haloacetamides; (iii) aldehydes, ketones,carboxyl, and maleimide groups.

Alternatively, a fusion protein comprising the antibody and cytotoxicagent may be made, e.g., by recombinant techniques or peptide synthesis.The length of DNA may comprise respective regions encoding the twoportions of the conjugate either adjacent one another or separated by aregion encoding a linker peptide which does not destroy the desiredproperties of the conjugate.

In yet another embodiment, the antibody may be conjugated to a“receptor” (such streptavidin) for utilization in tumor pre-targetingwherein the antibody-receptor conjugate is administered to theindividual, followed by removal of unbound conjugate from thecirculation using a clearing agent and then administration of a “ligand”(e.g., avidin) which is conjugated to a cytotoxic agent (e.g., aradionucleotide).

Methods Using Anti-FGFR3 Antibodies

The present invention features the use of an FGFR3 antibody as part of aspecific treatment regimen intended to provide a beneficial effect fromthe activity of this therapeutic agent. The present invention isparticularly useful in treating cancers of various types at variousstages.

The term cancer embraces a collection of proliferative disorders,including but not limited to pre-cancerous growths, benign tumors, andmalignant tumors. Benign tumors remain localized at the site of originand do not have the capacity to infiltrate, invade, or metastasize todistant sites. Malignant tumors will invade and damage other tissuesaround them. They can also gain the ability to break off from theoriginal site and spread to other parts of the body (metastasize),usually through the bloodstream or through the lymphatic system wherethe lymph nodes are located. Primary tumors are classified by the typeof tissue from which they arise; metastatic tumors are classified by thetissue type from which the cancer cells are derived. Over time, thecells of a malignant tumor become more abnormal and appear less likenormal cells. This change in the appearance of cancer cells is calledthe tumor grade, and cancer cells are described as beingwell-differentiated (low grade), moderately-differentiated,poorly-differentiated, or undifferentiated (high grade).Well-differentiated cells are quite normal appearing and resemble thenormal cells from which they originated. Undifferentiated cells arecells that have become so abnormal that it is no longer possible todetermine the origin of the cells.

Cancer staging systems describe how far the cancer has spreadanatomically and attempt to put patients with similar prognosis andtreatment in the same staging group. Several tests may be performed tohelp stage cancer including biopsy and certain imaging tests such as achest x-ray, mammogram, bone scan, CT scan, and MRI scan. Blood testsand a clinical evaluation are also used to evaluate a patient's overallhealth and detect whether the cancer has spread to certain organs.

To stage cancer, the American Joint Committee on Cancer first places thecancer, particularly solid tumors, in a letter category using the TNMclassification system. Cancers are designated the letter T (tumor size),N (palpable nodes), and/or M (metastases). T1, T2, T3, and T4 describethe increasing size of the primary lesion; NO, Ni, N2, N3 indicatesprogressively advancing node involvement; and M0 and M1 reflect theabsence or presence of distant metastases.

In the second staging method, also known as the Overall Stage Groupingor Roman Numeral Staging, cancers are divided into stages 0 to IV,incorporating the size of primary lesions as well as the presence ofnodal spread and of distant metastases. In this system, cases aregrouped into four stages denoted by Roman numerals I through IV, or areclassified as “recurrent.” For some cancers, stage 0 is referred to as“in situ” or “Tis,” such as ductal carcinoma in situ or lobularcarcinoma in situ for breast cancers. High grade adenomas can also beclassified as stage 0. In general, stage I cancers are small localizedcancers that are usually curable, while stage IV usually representsinoperable or metastatic cancer. Stage II and III cancers are usuallylocally advanced and/or exhibit involvement of local lymph nodes. Ingeneral, the higher stage numbers indicate more extensive disease,including greater tumor size and/or spread of the cancer to nearby lymphnodes and/or organs adjacent to the primary tumor. These stages aredefined precisely, but the definition is different for each kind ofcancer and is known to the skilled artisan.

Many cancer registries, such as the NCI's Surveillance, Epidemiology,and End Results Program (SEER), use summary staging. This system is usedfor all types of cancer. It groups cancer cases into five maincategories:

In situ is early cancer that is present only in the layer of cells inwhich it began.

Localized is cancer that is limited to the organ in which it began,without evidence of spread.

Regional is cancer that has spread beyond the original (primary) site tonearby lymph nodes or organs and tissues.

Distant is cancer that has spread from the primary site to distantorgans or distant lymph nodes.

Unknown is used to describe cases for which there is not enoughinformation to indicate a stage.

In addition, it is common for cancer to return months or years after theprimary tumor has been removed. Cancer that recurs after all visibletumor has been eradicated, is called recurrent disease. Disease thatrecurs in the area of the primary tumor is locally recurrent, anddisease that recurs as metastases is referred to as a distantrecurrence.

The tumor can be a solid tumor or a non-solid or soft tissue tumor.Examples of soft tissue tumors include leukemia (e.g., chronicmyelogenous leukemia, acute myelogenous leukemia, adult acutelymphoblastic leukemia, acute myelogenous leukemia, mature B-cell acutelymphoblastic leukemia, chronic lymphocytic leukemia, polymphocyticleukemia, or hairy cell leukemia) or lymphoma (e.g., non-Hodgkin'slymphoma, cutaneous T-cell lymphoma, or Hodgkin's disease). A solidtumor includes any cancer of body tissues other than blood, bone marrow,or the lymphatic system. Solid tumors can be further divided into thoseof epithelial cell origin and those of non-epithelial cell origin.Examples of epithelial cell solid tumors include tumors of thegastrointestinal tract, colon, breast, prostate, lung, kidney, liver,pancreas, ovary, head and neck, oral cavity, stomach, duodenum, smallintestine, large intestine, anus, gall bladder, labium, nasopharynx,skin, uterus, male genital organ, urinary organs, bladder, and skin.Solid tumors of non-epithelial origin include sarcomas, brain tumors,and bone tumors. Other examples of tumors are described in theDefinitions section.

In some embodiments, the patient herein is subjected to a diagnostictest e.g., prior to and/or during and/or after therapy. Generally, if adiagnostic test is performed, a sample may be obtained from a patient inneed of therapy. Where the subject has cancer, the sample may be a tumorsample, or other biological sample, such as a biological fluid,including, without limitation, blood, urine, saliva, ascites fluid, orderivatives such as blood serum and blood plasma, and the like.

The biological sample herein may be a fixed sample, e.g. a formalinfixed, paraffin-embedded (FFPE) sample, or a frozen sample.

Various methods for determining expression of mRNA or protein include,but are not limited to, gene expression profiling, polymerase chainreaction (PCR) including quantitative real time PCR (qRT-PCR),microarray analysis, serial analysis of gene expression (SAGE),MassARRAY, Gene Expression Analysis by Massively Parallel SignatureSequencing (MPSS), proteomics, immunohistochemistry (IHC), etc.Preferably mRNA is quantified. Such mRNA analysis is preferablyperformed using the technique of polymerase chain reaction (PCR), or bymicroarray analysis. Where PCR is employed, a preferred form of PCR isquantitative real time PCR (qRT-PCR). In one embodiment, expression ofone or more of the above noted genes is deemed positive expression if itis at the median or above, e.g. compared to other samples of the sametumor-type. The median expression level can be determined essentiallycontemporaneously with measuring gene expression, or may have beendetermined previously.

The steps of a representative protocol for profiling gene expressionusing fixed, paraffin-embedded tissues as the RNA source, including mRNAisolation, purification, primer extension and amplification are given invarious published journal articles (for example: Godfrey et al. J.Molec. Diagnostics 2: 84-91 (2000); Specht et al., Am. J. Pathol. 158:419-29 (2001)). Briefly, a representative process starts with cuttingabout 10 microgram thick sections of paraffin-embedded tumor tissuesamples. The RNA is then extracted, and protein and DNA are removed.After analysis of the RNA concentration, RNA repair and/or amplificationsteps may be included, if necessary, and RNA is reverse transcribedusing gene specific promoters followed by PCR. Finally, the data areanalyzed to identify the best treatment option(s) available to thepatient on the basis of the characteristic gene expression patternidentified in the tumor sample examined.

Detection of gene or protein expression may be determined directly orindirectly.

One may determine expression or translocation or amplification of FGFR3in the cancer (directly or indirectly). Various diagnostic/prognosticassays are available for this. In one embodiment, FGFR3 overexpressionmay be analyzed by IHC. Parafin embedded tissue sections from a tumorbiopsy may be subjected to the IHC assay and accorded a FGFR3 proteinstaining intensity criteria as follows:

Score 0 no staining is observed or membrane staining is observed in lessthan 10% of tumor cells.

Score 1+ a faint/barely perceptible membrane staining is detected inmore than 10% of the tumor cells. The cells are only stained in part oftheir membrane.

Score 2+ a weak to moderate complete membrane staining is observed inmore than 10% of the tumor cells.

Score 3+ a moderate to strong complete membrane staining is observed inmore than 10% of the tumor cells.

In some embodiments, those tumors with 0 or 1+ scores for FGFR3overexpression assessment may be characterized as not overexpressingFGFR3, whereas those tumors with 2+ or 3+ scores may be characterized asoverexpressing FGFR3.

In some embodiments, tumors overexpressing FGFR3 may be rated byimmunohistochemical scores corresponding to the number of copies ofFGFR3 molecules expressed per cell, and can been determinedbiochemically:

0=0-90 copies/cell,

1+=at least about 100 copies/cell,

2+=at least about 1000 copies/cell,

3+=at least about 10,000 copies/cell.

Alternatively, or additionally, FISH assays may be carried out onformalin-fixed, paraffin-embedded tumor tissue to determine the presenceor and/or extent (if any) of FGFR3 amplification or translocation in thetumor.

FGFR3 activation may be determined directly (e.g., by phospho-ELISAtesting, or other means of detecting phosphorylated receptor) orindirectly (e.g., by detection of activated downstream signaling pathwaycomponents, detection of receptor dimers (e.g., homodimers,heterodimers), detection of gene expression profiles and the like.

Similarly, constitutive FGFR3 and/or ligand-independent orligand-dependent FGFR3 may be detected directly or indirectly (e.g., bydetection of receptor mutations correlated with constitutive activity,by detection of receptor amplification correlated with constitutiveactivity and the like).

Methods for detection of nucleic acid mutations are well known in theart. Often, though not necessarily, a target nucleic acid in a sample isamplified to provide the desired amount of material for determination ofwhether a mutation is present. Amplification techniques are well knownin the art. For example, the amplified product may or may not encompassall of the nucleic acid sequence encoding the protein of interest, solong as the amplified product comprises the particular aminoacid/nucleic acid sequence position where the mutation is suspected tobe.

In one example, presence of a mutation can be determined by contactingnucleic acid from a sample with a nucleic acid probe that is capable ofspecifically hybridizing to nucleic acid encoding a mutated nucleicacid, and detecting said hybridization. In one embodiment, the probe isdetectably labeled, for example with a radioisotope (³H, ³²P, ³³P etc),a fluorescent agent (rhodamine, fluorescene etc.) or a chromogenicagent. In some embodiments, the probe is an antisense oligomer, forexample PNA, morpholino-phosphoramidates, LNA or 2′-alkoxyalkoxy. Theprobe may be from about 8 nucleotides to about 100 nucleotides, or about10 to about 75, or about 15 to about 50, or about 20 to about 30. Inanother aspect, nucleic acid probes of the invention are provided in akit for identifying FGFR3 mutations in a sample, said kit comprising anoligonucleotide that specifically hybridizes to or adjacent to a site ofmutation in the nucleic acid encoding FGFR3. The kit may furthercomprise instructions for treating patients having tumors that containFGFR3 mutations with a FGFR3 antagonist based on the result of ahybridization test using the kit.

Mutations can also be detected by comparing the electrophoretic mobilityof an amplified nucleic acid to the electrophoretic mobility ofcorresponding nucleic acid encoding wild-type FGFR3. A difference in themobility indicates the presence of a mutation in the amplified nucleicacid sequence. Electrophoretic mobility may be determined by anyappropriate molecular separation technique, for example on apolyacrylamide gel.

Nucleic acids may also be analyzed for detection of mutations usingEnzymatic Mutation Detection (EMD) (Del Tito et al, Clinical Chemistry44:731-739, 1998). EMD uses the bacteriophage resolvase T4 endonucleaseVII, which scans along double-stranded DNA until it detects and cleavesstructural distortions caused by base pair mismatches resulting fromnucleic acid alterations such as point mutations, insertions anddeletions. Detection of two short fragments formed by resolvasecleavage, for example by gel electrophoresis, indicates the presence ofa mutation. Benefits of the EMD method are a single protocol to identifypoint mutations, deletions, and insertions assayed directly fromamplification reactions, eliminating the need for sample purification,shortening the hybridization time, and increasing the signal-to-noiseratio. Mixed samples containing up to a 20-fold excess of normal nucleicacids and fragments up to 4 kb in size can been assayed. However, EMDscanning does not identify particular base changes that occur inmutation positive samples, therefore often requiring additionalsequencing procedures to identify the specific mutation if necessary.CEL I enzyme can be used similarly to resolvase T4 endonuclease VII, asdemonstrated in U.S. Pat. No. 5,869,245.

Another simple kit for detecting mutations is a reverse hybridizationtest strip similar to Haemochromatosis StripAssay™ (Viennalabshttp://www.bamburghmarrsh.com/pdf/4220.pdf) for detection of multiplemutations in HFE, TFR2 and FPN1 genes causing Haemochromatosis. Such anassay is based on sequence specific hybridization followingamplification by PCR. For single mutation assays, a microplate-baseddetection system may be applied, whereas for multi-mutation assays, teststrips may be used as “macro-arrays”. Kits may include ready-to usereagents for sample prep, amplification and mutation detection.Multiplex amplification protocols provide convenience and allow testingof samples with very limited volumes. Using the straightforwardStripAssay format, testing for twenty and more mutations may becompleted in less than five hours without costly equipment. DNA isisolated from a sample and the target nucleic acid is amplified in vitro(e.g., by PCR) and biotin-labelled, generally in a single (“multiplex”)amplification reaction. The amplification products are then selectivelyhybridized to oligonucleotide probes (wild-type and mutant specific)immobilized on a solid support such as a test strip in which the probesare immobilized as parallel lines or bands. Bound biotinylated ampliconsare detected using streptavidin-alkaline phosphatase and colorsubstrates. Such an assay can detect all or any subset of the mutationsof the invention. With respect to a particular mutant probe band, one ofthree signaling patterns are possible: (i) a band only for wild-typeprobe which indicates normal nucleic acid sequence, (ii) bands for bothwild-type and a mutant probe which indicates heterozygous genotype, and(iii) band only for the mutant probe which indicates homozygous mutantgenotype. Accordingly, in one aspect, the invention provides a method ofdetecting mutations of the invention comprising isolating and/oramplifying a target FGFR3 nucleic acid sequence from a sample, such thatthe amplification product comprises a ligand, contacting theamplification product with a probe which comprises a detectable bindingpartner to the ligand and the probe is capable of specificallyhydribizing to a mutation of the invention, and then detecting thehybridization of said probe to said amplification product. In oneembodiment, the ligand is biotin and the binding partner comprisesavidin or streptavidin. In one embodiment, the binding partner comprisessteptavidin-alkaline which is detectable with color substrates. In oneembodiment, the probes are immobilized for example on a test stripwherein probes complementary to different mutations are separated fromone another.

Alternatively, the amplified nucleic acid is labelled with aradioisotope in which case the probe need not comprise a detectablelabel.

Alterations of a wild-type gene encompass all forms of mutations such asinsertions, inversions, deletions, and/or point mutations. In oneembodiment, the mutations are somatic. Somatic mutations are those whichoccur only in certain tissues, e.g., in the tumor tissue, and are notinherited in the germ line. Germ line mutations can be found in any of abody's tissues.

A sample comprising a target nucleic acid can be obtained by methodswell known in the art, and that are appropriate for the particular typeand location of the tumor. Tissue biopsy is often used to obtain arepresentative piece of tumor tissue. Alternatively, tumor cells can beobtained indirectly in the form of tissues/fluids that are known orthought to contain the tumor cells of interest. For instance, samples oflung cancer lesions may be obtained by resection, bronchoscopy, fineneedle aspiration, bronchial brushings, or from sputum, pleural fluid orblood. Mutant genes or gene products can be detected from tumor or fromother body samples such as urine, sputum or serum. The same techniquesdiscussed above for detection of mutant target genes or gene products intumor samples can be applied to other body samples. Cancer cells aresloughed off from tumors and appear in such body samples. By screeningsuch body samples, a simple early diagnosis can be achieved for diseasessuch as cancer. In addition, the progress of therapy can be monitoredmore easily by testing such body samples for mutant target genes or geneproducts.

Means for enriching a tissue preparation for tumor cells are known inthe art. For example, the tissue may be isolated from paraffin orcryostat sections. Cancer cells may also be separated from normal cellsby flow cytometry or laser capture microdissection. These, as well asother techniques for separating tumor from normal cells, are well knownin the art. If the tumor tissue is highly contaminated with normalcells, detection of mutations may be more difficult, although techniquesfor minimizing contamination and/or false positive/negative results areknown, some of which are described hereinbelow. For example, a samplemay also be assessed for the presence of a biomarker (including amutation) known to be associated with a tumor cell of interest but not acorresponding normal cell, or vice versa.

Detection of point mutations in target nucleic acids may be accomplishedby molecular cloning of the target nucleic acids and sequencing thenucleic acids using techniques well known in the art. Alternatively,amplification techniques such as the polymerase chain reaction (PCR) canbe used to amplify target nucleic acid sequences directly from a genomicDNA preparation from the tumor tissue. The nucleic acid sequence of theamplified sequences can then be determined and mutations identifiedtherefrom. Amplification techniques are well known in the art, e.g.,polymerase chain reaction as described in Saiki et al., Science 239:487,1988; U.S. Pat. Nos. 4,683,203 and 4,683,195.

It should be noted that design and selection of appropriate primers arewell established techniques in the art.

The ligase chain reaction, which is known in the art, can also be usedto amplify target nucleic acid sequences. See, e.g., Wu et al.,Genomics, Vol. 4, pp. 560-569 (1989). In addition, a technique known asallele specific PCR can also be used. See, e.g., Ruano and Kidd, NucleicAcids Research, Vol. 17, p. 8392, 1989. According to this technique,primers are used which hybridize at their 3′ ends to a particular targetnucleic acid mutation. If the particular mutation is not present, anamplification product is not observed. Amplification Refractory MutationSystem (ARMS) can also be used, as disclosed in European PatentApplication Publication No. 0332435, and in Newton et al., Nucleic AcidsResearch, Vol. 17, p.7, 1989. Insertions and deletions of genes can alsobe detected by cloning, sequencing and amplification. In addition,restriction fragment length polymorphism (RFLP) probes for the gene orsurrounding marker genes can be used to score alteration of an allele oran insertion in a polymorphic fragment. Single stranded conformationpolymorphism (SSCP) analysis can also be used to detect base changevariants of an allele. See, e.g. Orita et al., Proc. Natl. Acad. Sci.USA Vol. 86, pp. 2766-2770, 1989, and Genomics, Vol. 5, pp. 874-879,1989. Other techniques for detecting insertions and deletions as knownin the art can also be used.

Alteration of wild-type genes can also be detected on the basis of thealteration of a wild-type expression product of the gene. Suchexpression products include both mRNA as well as the protein product.Point mutations may be detected by amplifying and sequencing the mRNA orvia molecular cloning of cDNA made from the mRNA. The sequence of thecloned cDNA can be determined using DNA sequencing techniques which arewell known in the art. The cDNA can also be sequenced via the polymerasechain reaction (PCR).

Mismatches are hybridized nucleic acid duplexes which are not 100%complementary. The lack of total complementarity may be due todeletions, insertions, inversions, substitutions or frameshiftmutations. Mismatch detection can be used to detect point mutations in atarget nucleic acid. While these techniques can be less sensitive thansequencing, they are simpler to perform on a large number of tissuesamples. An example of a mismatch cleavage technique is the RNaseprotection method, which is described in detail in Winter et al., Proc.Natl. Acad. Sci. USA, Vol. 82, p. 7575, 1985, and Meyers et al.,Science, Vol. 230, p. 1242, 1985. For example, a method of the inventionmay involve the use of a labeled riboprobe which is complementary to thehuman wild-type target nucleic acid. The riboprobe and target nucleicacid derived from the tissue sample are annealed (hybridized) togetherand subsequently digested with the enzyme RNase A which is able todetect some mismatches in a duplex RNA structure. If a mismatch isdetected by RNase A, it cleaves at the site of the mismatch. Thus, whenthe annealed RNA preparation is separated on an electrophoretic gelmatrix, if a mismatch has been detected and cleaved by RNase A, an RNAproduct will be seen which is smaller than the full-length duplex RNAfor the riboprobe and the mRNA or DNA. The riboprobe need not be thefull length of the target nucleic acid mRNA or gene, but can a portionof the target nucleic acid, provided it encompasses the positionsuspected of being mutated. If the riboprobe comprises only a segment ofthe target nucleic acid mRNA or gene, it may be desirable to use anumber of these probes to screen the whole target nucleic acid sequencefor mismatches if desired.

In a similar manner, DNA probes can be used to detect mismatches, forexample through enzymatic or chemical cleavage. See, e.g., Cotton etal., Proc. Natl. Acad. Sci. USA, Vol. 85, 4397, 1988; and Shenk et al.,Proc. Natl. Acad. Sci. USA, Vol. 72, p. 989, 1975. Alternatively,mismatches can be detected by shifts in the electrophoretic mobility ofmismatched duplexes relative to matched duplexes. See, e.g., Cariello,Human Genetics, Vol. 42, p. 726, 1988. With either riboprobes or DNAprobes, the target nucleic acid mRNA or DNA which might contain amutation can be amplified before hybridization. Changes in targetnucleic acid DNA can also be detected using Southern hybridization,especially if the changes are gross rearrangements, such as deletionsand insertions.

Target nucleic acid DNA sequences which have been amplified may also bescreened using allele-specific probes. These probes are nucleic acidoligomers, each of which contains a region of the target nucleic acidgene harboring a known mutation. For example, one oligomer may be about30 nucleotides in length, corresponding to a portion of the target genesequence. By use of a battery of such allele-specific probes, targetnucleic acid amplification products can be screened to identify thepresence of a previously identified mutation in the target gene.Hybridization of allele-specific probes with amplified target nucleicacid sequences can be performed, for example, on a nylon filter.Hybridization to a particular probe under stringent hybridizationconditions indicates the presence of the same mutation in the tumortissue as in the allele-specific probe.

Alteration of wild-type target genes can also be detected by screeningfor alteration of the corresponding wild-type protein. For example,monoclonal antibodies immunoreactive with a target gene product can beused to screen a tissue, for example an antibody that is known to bindto a particular mutated position of the gene product (protein). Forexample, an antibody that is used may be one that binds to a deletedexon or that binds to a conformational epitope comprising a deletedportion of the target protein. Lack of cognate antigen would indicate amutation. Antibodies specific for products of mutant alleles could alsobe used to detect mutant gene product. Antibodies may be identified fromphage display libraries. Such immunological assays can be done in anyconvenient format known in the art. These include Western blots,immunohistochemical assays and ELISA assays. Any means for detecting analtered protein can be used to detect alteration of wild-type targetgenes.

Primer pairs are useful for determination of the nucleotide sequence ofa target nucleic acid using nucleic acid amplification techniques suchas the polymerase chain reaction. The pairs of single stranded DNAprimers can be annealed to sequences within or surrounding the targetnucleic acid sequence in order to prime amplification of the targetsequence. Allele-specific primers can also be used. Such primers annealonly to particular mutant target sequence, and thus will only amplify aproduct in the presence of the mutant target sequence as a template. Inorder to facilitate subsequent cloning of amplified sequences, primersmay have restriction enzyme site sequences appended to their ends. Suchenzymes and sites are well known in the art. The primers themselves canbe synthesized using techniques which are well known in the art.Generally, the primers can be made using oligonucleotide synthesizingmachines which are commercially available. Design of particular primersis well within the skill of the art.

Nucleic acid probes are useful for a number of purposes. They can beused in Southern hybridization to genomic DNA and in the RNaseprotection method for detecting point mutations already discussed above.The probes can be used to detect target nucleic acid amplificationproducts. They may also be used to detect mismatches with the wild typegene or mRNA using other techniques. Mismatches can be detected usingeither enzymes (e.g., S1 nuclease), chemicals (e.g., hydroxylamine orosmium tetroxide and piperidine), or changes in electrophoretic mobilityof mismatched hybrids as compared to totally matched hybrids. Thesetechniques are known in the art. See Novack et al., Proc. Natl. Acad.Sci. USA, Vol. 83, p. 586, 1986. Generally, the probes are complementaryto sequences outside of the kinase domain. An entire battery of nucleicacid probes may be used to compose a kit for detecting mutations intarget nucleic acids. The kit allows for hybridization to a large regionof a target sequence of interest. The probes may overlap with each otheror be contiguous.

If a riboprobe is used to detect mismatches with mRNA, it is generallycomplementary to the mRNA of the target gene. The riboprobe thus is anantisense probe in that it does not code for the corresponding geneproduct because it is complementary to the sense strand. The riboprobegenerally will be labeled with a radioactive, colorimetric, orfluorometric material, which can be accomplished by any means known inthe art. If the riboprobe is used to detect mismatches with DNA it canbe of either polarity, sense or anti-sense. Similarly, DNA probes alsomay be used to detect mismatches.

In some instances, the cancer does or does not overexpress FGFR3.Receptor overexpression may be determined in a diagnostic or prognosticassay by evaluating increased levels of the receptor protein present onthe surface of a cell (e.g. via an immunohistochemistry assay; IHC).Alternatively, or additionally, one may measure levels ofreceptor-encoding nucleic acid in the cell, e.g. via fluorescent in situhybridization (FISH; see WO98/45479 published October, 1998), southernblotting, or polymerase chain reaction (PCR) techniques, such as realtime quantitative PCR (RT-PCR). Aside from the above assays, various invivo assays are available to the skilled practitioner. For example, onemay expose cells within the body of the patient to an antibody which isoptionally labeled with a detectable label, e.g. a radioactive isotope,and binding of the antibody to cells in the patient can be evaluated,e.g. by external scanning for radioactivity or by analyzing a biopsytaken from a patient previously exposed to the antibody.

Chemotherapeutic Agents

The combination therapy of the invention can further comprise one ormore chemotherapeutic agent(s). The combined administration includescoadministration or concurrent administration, using separateformulations or a single pharmaceutical formulation, and consecutiveadministration in either order, wherein preferably there is a timeperiod while both (or all) active agents simultaneously exert theirbiological activities.

The chemotherapeutic agent, if administered, is usually administered atdosages known therefor, or optionally lowered due to combined action ofthe drugs or negative side effects attributable to administration of theantimetabolite chemotherapeutic agent. Preparation and dosing schedulesfor such chemotherapeutic agents may be used according to manufacturers'instructions or as determined empirically by the skilled practitioner.

Various chemotherapeutic agents that can be combined are disclosedherein.

In some embodiments, chemotherapeutic agents to be combined are selectedfrom the group consisting of lenalidomide (REVLIMID), proteosomeinhibitors (such as bortezomib (VELCADE) and PS342), bora taxoid(including docetaxel and paclitaxel), vinca (such as vinorelbine orvinblastine), platinum compound (such as carboplatin or cisplatin),aromatase inhibitor (such as letrozole, anastrazole, or exemestane),anti-estrogen (e.g. fulvestrant or tamoxifen), etoposide, thiotepa,cyclophosphamide, pemetrexed, methotrexate, liposomal doxorubicin,pegylated liposomal doxorubicin, capecitabine, gemcitabine, melthalin,doxorubicin, vincristine, COX-2 inhibitor (for instance, celecoxib), orsteroid (e.g., dexamethasone and prednisone). In some embodiments (e.g.,embodiments involving treatment of t(4; 14)+ multiple myeloma,dexamethasone and lenalidomide, or dexamethasone, or bortezomib, orvincristine, doxorubicin and dexamethason, or thalidomide anddexamethasone, or liposomal doxorubicin, vincristine and dexamethasone,or lenalidomide and dexamethasone, or bortezomib and dexamethasone, orbortezomib, doxorubicin, and dexamethasone are combined. In someembodiments (e.g., embodiments involving bladder cancer), gemcitabineand cisplatin, or a taxane (e.g., paclitaxel, docetaxel), or pemetrexed,or methotrexate, vinblastine, doxorubicin and cisplatin, or carboplatin,or mitomycin C in combination with 5-Fluorouracil, or cisplatin, orcisplatin and 5-Fluorouracil are combined.

Formulations, Dosages and Administrations

The therapeutic agents used in the invention will be formulated, dosed,and administered in a fashion consistent with good medical practice.Factors for consideration in this context include the particulardisorder being treated, the particular subject being treated, theclinical condition of the individual patient, the cause of the disorder,the site of delivery of the agent, the method of administration, thescheduling of administration, the drug-drug interaction of the agents tobe combined, and other factors known to medical practitioners.

Therapeutic formulations are prepared using standard methods known inthe art by mixing the active ingredient having the desired degree ofpurity with optional physiologically acceptable carriers, excipients orstabilizers (Remington's Pharmaceutical Sciences (20^(th) edition), ed.A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.).Acceptable carriers, include saline, or buffers such as phosphate,citrate and other organic acids; antioxidants including ascorbic acid;low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilicpolymers such as polyvinylpyrrolidone, amino acids such as glycine,glutamine, asparagines, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counterions such as sodium; and/or nonionicsurfactants such as TWEEN™, PLURONICS™, or PEG.

Optionally, but preferably, the formulation contains a pharmaceuticallyacceptable salt, preferably sodium chloride, and preferably at aboutphysiological concentrations. Optionally, the formulations of theinvention can contain a pharmaceutically acceptable preservative. Insome embodiments the preservative concentration ranges from 0.1 to 2.0%,typically v/v. Suitable preservatives include those known in thepharmaceutical arts. Benzyl alcohol, phenol, m-cresol, methylparaben,and propylparaben are preferred preservatives. Optionally, theformulations of the invention can include a pharmaceutically acceptablesurfactant at a concentration of 0.005 to 0.02%.

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Such molecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, supra.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsule. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS-S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

The therapeutic agents of the invention are administered to a humanpatient, in accord with known methods, such as intravenousadministration as a bolus or by continuous infusion over a period oftime, by intramuscular, intraperitoneal, intracerobrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. An ex vivo strategy can also be used fortherapeutic applications. Ex vivo strategies involve transfecting ortransducing cells obtained from the subject with a polynucleotideencoding a FGFR3 antagonist. The transfected or transduced cells arethen returned to the subject. The cells can be any of a wide range oftypes including, without limitation, hemopoietic cells (e.g., bonemarrow cells, macrophages, monocytes, dendritic cells, T cells, or Bcells), fibroblasts, epithelial cells, endothelial cells, keratinocytes,or muscle cells.

For example, if the FGFR3 antagonist is an antibody, the antibody isadministered by any suitable means, including parenteral, subcutaneous,intraperitoneal, intrapulmonary, and intranasal, and, if desired forlocal immunosuppressive treatment, intralesional administration.Parenteral infusions include intramuscular, intravenous, intraarterial,intraperitoneal, or subcutaneous administration. In addition, theantibody is suitably administered by pulse infusion, particularly withdeclining doses of the antibody. Preferably the dosing is given byinjections, most preferably intravenous or subcutaneous injections,depending in part on whether the administration is brief or chronic.

In another example, the FGFR3 antagonist compound is administeredlocally, e.g., by direct injections, when the disorder or location ofthe tumor permits, and the injections can be repeated periodically. TheFGFR3 antagonist can also be delivered systemically to the subject ordirectly to the tumor cells, e.g., to a tumor or a tumor bed followingsurgical excision of the tumor, in order to prevent or reduce localrecurrence or metastasis.

Administration of the therapeutic agents in combination typically iscarried out over a defined time period (usually minutes, hours, days orweeks depending upon the combination selected). Combination therapy isintended to embrace administration of these therapeutic agents in asequential manner, that is, wherein each therapeutic agent isadministered at a different time, as well as administration of thesetherapeutic agents, or at least two of the therapeutic agents, in asubstantially simultaneous manner.

The therapeutic agent can be administered by the same route or bydifferent routes. For example, the anti-FGFR3 antibody in thecombination may be administered by intravenous injection while achemotherapeutic agent in the combination may be administered orally.Alternatively, for example, both of the therapeutic agents may beadministered orally, or both therapeutic agents may be administered byintravenous injection, depending on the specific therapeutic agents. Thesequence in which the therapeutic agents are administered also variesdepending on the specific agents.

Depending on the type and severity of the disease, about 1 μg/kg to 100mg/kg of each therapeutic agent is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. A typical dailydosage might range from about 1 μg/kg to about 100 mg/kg or more,depending on the factors mentioned above. For repeated administrationsover several days or longer, depending on the condition, the treatmentis sustained until the cancer is treated, as measured by the methodsdescribed above. However, other dosage regimens may be useful.

The present application contemplates administration of the FGFR3antibody by gene therapy. See, for example, WO96/07321 published Mar.14, 1996 concerning the use of gene therapy to generate intracellularantibodies.

Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, etc. The containers may be formed from a variety ofmaterials such as glass or plastic. The container holds a compositionwhich is by itself or when combined with another composition(s)effective for treating, preventing and/or diagnosing the condition andmay have a sterile access port (for example the container may be anintravenous solution bag or a vial having a stopper pierceable by ahypodermic injection needle). At least one active agent in thecomposition is an antibody of the invention. The label or package insertindicates that the composition is used for treating the condition ofchoice, such as cancer. Moreover, the article of manufacture maycomprise (a) a first container with a composition contained therein,wherein the composition comprises an antibody of the invention; and (b)a second container with a composition contained therein, wherein thecomposition comprises a further cytotoxic agent. The article ofmanufacture in this embodiment of the invention may further comprise apackage insert indicating that the first and second antibodycompositions can be used to treat a particular condition, e.g., cancer.Alternatively, or additionally, the article of manufacture may furthercomprise a second (or third) container comprising apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

The following are examples of the methods and compositions of theinvention. It is understood that various other embodiments may bepracticed, given the general description provided above.

Examples Materials and Methods Cell Lines and Cell Culture

The cell line RT4 was obtained from American Type Cell CultureCollection. Cell lines RT112, OPM2 and Ba/F3 were purchased from GermanCollection of Microorganisms and Cell Cultures (DSMZ, (Germany)).Multiple myeloma cell line KMS11 was kindly provided by Dr. TakemiOtsuki at Kawasaki Medical School (Japan). Bladder cancer cell lineTCC-97-7 was a generous gift from Dr. Margaret Knowles at St James'sUniversity Hospital (Leeds, UK). UMUC-14 cell line was obtained from Dr.H. B. Grossman (currently at University of Texas M.D. Anderson CancerCenter, TX). The cells were maintained with RPMI medium supplementedwith 10% fetal bovine serum (FBS) (Sigma), 100 U/ml penicillin, 0.1mg/ml streptomycin and L-glutamine under conditions of 5% CO₂ at 37° C.

FGFR3^(S249C) Dimerization Studies

UMUC-14 cells were grown in cysteine-free medium, treated with R3Mab orDTNB for 3 hr, and cell lysates were subject to immunoblot analysisunder reducing or non-reducing conditions. For in vitro dimerizationstudies, FGFR3-IIIb^(S249C) (residues 143-374) was cloned into pAcGP67Avector and expressed in T. ni Pro cells. The recombinant protein waspurified through Ni-NTA column followed by Superdex S200 column. DimericFGFR3^(S249C) was eluted in 25 mM Tris (pH 7.5) and 300 mM NaCl. R3Mab(1 μM) was incubated with FGFR3^(S249C) dimer (0.1 μM) at 37° C. underthe following conditions: 100 mM KH₂PO4 (pH 7.5), 25 μM DTT, 1 mM EDTAand 0.75 mg/ml BSA. Aliquots of the reaction were taken at indicatedtime points and the reaction was stopped by adding sample buffer withoutβ-mercaptoethanol. Dimer-monomer was analyzed by immunoblot.

Xenograft Studies

All studies were approved by Genentech's Institutional Animal Care andUse Committee. Female nu/nu mice or CB 17 severe combinedimmunodeficiency (SCID) mice, 6-8 weeks of age, were purchased fromCharles River Laboratory (Hollister, Calif.). Female athymic nude micewere obtained from the National Cancer Institute-Frederick CancerCenter. Mice were maintained under specific pathogen-free conditions.RT112 shRNA stable cells (7×10⁶), RT112 (7×10⁶), Ba/F3-FGFR3^(S249C)(5×10⁶), OPM2 (15×10⁶), or KMS11 cells (20×10⁶) were implantedsubcutaneously into the flank of mice in a volume of 0.2 ml inHBSS/matrigel (1:1 v/v, BD Biosciences). UMUC-14 cells (5×10⁶) wereimplanted without matrigel. Tumors were measured twice weekly using acaliper, and tumor volume was calculated using the formula: V=0.5 a×b²,where a and b are the length and width of the tumor, respectively. Whenthe mean tumor volume reached 150-200 mm³, mice were randomized intogroups of 10 and were treated twice weekly with intraperitoneal (i.p)injection of R3Mab (0.3-50 mg/kg), or a control human IgG1 diluted inHBSS. Control animals were given vehicle (HBSS) alone.

Statistics

Pooled data are expressed as mean+/−SEM. Unpaired Student's t tests(2-tailed) were used for comparison between two groups. A value ofP<0.05 was considered statistically significant in all experiments.

Generation of FGFR3 shRNA Stable Cells

Three independent FGFR3 shRNA were cloned into pHUSH vector as described(1). The sequence for FGFR3 shRNAs used in the studies is as follows:shRNA2: 5′GATCCCCGCATCAAGCTGCGGCATCATTCAAGAGATGATGCCGCAGCTTGATGCTTTTTTGGAAA (SEQ ID NO:192); shRNA4:5′-GATCCCCTGCACAACCTCGACTACTATTCAAGAGATAGTAGTCGAGGTTGTGCATTTTTT GGAAA-3′(SEQ ID NO:193); shRNA6:5′-GATCCCCAACCTCGACTACTACAAGATTCAAGAGATCTTGTAGTAGTCGAGGTTTTTTTT GGAAA-3′(SEQ ID NO: 194). All constructs were confirmed by sequencing. EGFPcontrol shRNA was described in our previous study (50). The shRNAcontaining retrovirus was produced by co-transfecting GP2-293 packagingcells (Clontech Laboratories, Mountain View, Calif.) with VSV-G(Clontech Laboratories) and pHUSH-FGFR3 shRNA constructs, and viralsupernatants were harvested 72 hr after transfection, and cleared ofcell debris by centrifugation for transduction experiment.

RT112 cells were maintained in RPMI 1640 medium containingtetracycline-free FBS (Clontech Laboratories), and transduced withretroviral supernatant in the presence of 4 μg/ml polybrene. 72 hoursafter infection, 2 μg/ml puromycin (Clontech Laboratories) was added tothe medium to select stable clones expressing shRNA. Stable cells wereisolated, treated with 0.1 or 1 μg/ml doxycycline (ClontechLaboratories) for 4 days, and inducible knockdown of FGFR3 proteinexpression was assessed by Western blotting analysis. Cell cycleanalyses were performed as described (51).

Selecting Phage Antibodies Specific for FGFR3

Human phage antibody libraries with synthetic diversities in theselected complementary determining regions (H1, H2, H3, L3), mimickingthe natural diversity of human IgG repertoire were used for panning. TheFab fragments were displayed bivalently on the surface of M13bacteriophage particles (52). His-tagged IgD2-D3 of human FGFR3-IIIb andIIIc were used as antigens. 96-well MaxiSorp immunoplates (Nunc) werecoated overnight at 4° C. with FGFR3-IIIb-His protein or FGFR3-IIIC-Hisprotein (10 μg/ml) and blocked for 1 hour with PBST buffer (PBS with0.05% Tween 20) supplemented with 1% BSA. The antibody phage librarieswere added and incubated overnight at room temperature (RT). The plateswere washed with PBST buffer and bound phage were eluted with 50 mM HCland 500 mM NaCl for 30 minutes and neutralized with equal volume of 1MTris base. Recovered phages were amplified in E. coli XL-1 blue cells.During subsequent selection rounds, the incubation time of the phageantibodies was decreased to 2 hours and the stringency of plate washingwas gradually increased (53). Unique and specific phage antibodies thatbind to both IIIb and IIIc isoforms of FGFR3 were identified by phageELISA and DNA sequencing. Out of 400 clones screened, four were selectedto reformat to full length IgGs by cloning VL and VH regions ofindividual clones into LPG3 and LPG4 vectors, respectively, transientlyexpressed in mammalian cells, and purified with protein A columns (54).Clone 184.6 was selected for affinity maturation.

For affinity maturation, phagemid displaying monovalent Fab on thesurface of M13 bacteriophage (52) served as the library template forgrafting light chain (VL) and heavy chain (VH) variable domains of thephage Ab. Stop codons was incorporated in CDR-L3. A soft randomizationstrategy was adopted for affinity maturation as described (53). Twodifferent combinations of CDR loops, H1/H2/L3, H3/L3, or L1/L2/L3 wereselected for randomization. For selecting affinity-matured clones, phagelibraries were sorted against FGFR3 IIIb or IIIc-His protein, subjectedto plate sorting for the first round and followed by four rounds ofsolution phase sorting as described (52). After five rounds of panning,a high-throughput single-point competitive phage ELISA was used torapidly screen for high-affinity clones as described (55). Clones withlow ratio of the absorbance at 450 nm in the presence of 10 nM FGFR3-Histo that in the absence of FGFR3-His were chosen for furthercharacterization.

Clones 184.6.1, 184.6.21, 184.6.49, 184.6.51, 184.6.58, 184.6.62 and184.6.92 significantly reduced viability of Ba/F3-FGFR3-IIIb,Ba/F3-FGFR3-IIIc and Ba/F3-FGFR3-S249C cell lines, and clone 184.6.52significantly reduced the viability of the Ba/F3-FGFR3-S249C cell line.The increased inhibitory activity ranged from about 50-fold (clone184.6.52) to about 100-fold (clones 184.6.1, 184.6.21, 184.6.49,184.6.51, 184.6.58, 184.6.62 and 184.6.92) greater than parent clone184.6, depending on the cell line assayed. Binding kinetics of clones184.6.1, 184.6.58, and 184.6.62 to FGFR3-IIIb and FGFR3-IIIc weredetermined using BIAcore as follows:

FGFR3-IIIb KD (M) FGFR3-IIIc KD (M) 184.6 3.80E−08 1.10E−07 184.6.12.64E−10 1.44E−09 184.6.58 1.90E−10 8.80E−10 184.6.62 1.20E−10 2.24E−09Clones 184.6.1, 184.6.58, and 184.6.62 also showed improved inhibitionof FGFR3 downstream signaling in Ba/F3-FGFR3 cells, RT112 cells and OPM2cells.

Clone 184.6.1 was selected. A sequence modification, N54S, wasintroduced into HVR H2 at residue 54, to improve manufacturability,creating clone 184.6.1N54S. Clones 184.6.1 and 184.6.1N54S displayedcomparable binding kinetics (measured in Biacore assays) and comparableactivity in the Ba/F3 cell viability assay. Additional HVR H2 variantswere generated: N54S was introduced in clone 184.6.58, and N54G, N54A,or N54Q were introduced in clone 184.6.1 and 184.6.58. These clonesshowed comparable activity in the Ba/F3 cell viability assay to parentclones 184.6.1 or 184.6.58.

Another sequence modification, D30E, was introduced into HVR L1 of clone184.6.1N54S, creating clone 184.6.1NSD30E. Clone 184.6.1NSD30E and clone184.6.1N54S showed comparable binding kinetics and comparable activityin the BA/F3 cell viability assay to parent clones 184.6.1 or 184.6.58.

As used herein, “R3 Mab” refers to anti-FGFR3 antibody clones184.6.1N54S, 184.6.1, or 184.6. Clone 184.6.1N54S was used in figuresand experiments referencing “R3Mab”, except in the experiments leadingto the results shown in the following figures (for which the antibodyused is shown in parentheses): FIGS. 9B (clone 184.6.1), 10A-F (clone184.6), 11A and 11B (clone 184.6), 13A-E (clone 184.6.1), 14A (clone184.6.1), 14B, 14G, and 14H (clone 184.6), 19A-E (clone184.6.1), and 22Band 22C (clone 184.6.1).

BIAcore/Surface Plasmon Resonance (SRP) Analysis to Determine AntibodyBinding Affinities

Binding affinities of R3Mab to FGFR3 were measured by Biacore/SRP usinga BIAcore™-3000 instrument as described (52) with the followingmodifications. R3Mab was directly coated on CM5 biosensor chips toachieve approximately 400 response units (RU). For kinetic measurement,two-fold serial dilutions of FGFR3-IIIb or IIIc-His protein (startingfrom 67 nM) were injected in PBST buffer at 25° C. with a flow rate of30 μl/minute. Association rates (Kon, per mol/s) and dissociation rates(Koff, per s) were calculated using a simple one-one Langmuir bindingmodel (BIAcore Evaluation Software version 3.2). The equilibriumdissociation constant (Kd, per mol) was calculated as the ratio ofKoff/Kon.

Binding affinities of mouse hybridoma antibodies to FGFR3 were measuredby Biacore/SRP as follows. Human FGFR3-IIIb or IIIc was coupled ontothree different flow cells (FC), FC2, FC3 and FC4, of a BIACORE™ CM5sensor chip to achieve the response unit (RU) about 50 RU.Immobilization was achieved by random coupling through amino groupsusing a protocol provided by the manufacturer. Sensorgrams were recordedfor binding of hybridoma-derived anti-FGFR3 murine IgG or the Fabfragment to these surfaces at 25° C. by injection of a series ofsolutions ranging from 250 nM to 0.48 nM in 2-fold increments at a flowrate of 30 μl/min. Between each injection, 10 mM Glycine-HCl pH 1.7 wasserved as the buffer to regenerate the sensor chip. The signal from thereference cell (FC1) was subtracted from the observed sensorgram at FC2,FC3 and FC4. Kinetic constants were calculated by nonlinear regressionfitting of the data according to a 1:1 Langmuir binding model usingBIAcore evaluation software (version 3.2) supplied by the manufacturer.

ELISA Binding Studies

cDNAs encoding the extracellular domains (ECD) of human FGFR1-IIIb,IIIc, FGFR2-IIIb and IIIc, FGFR3-IIIb and IIIc, and FGFR4 were clonedinto pRK-based vector to generate human FGFR-human Fc chimeric proteins.The recombinant proteins were produced by transiently transfectingChinese hamster ovary (CHO) cells and purified via protein A affinitychromatography. To test binding of antibodies to human FGFRs, Maxisorp96-well plates (Nunc) were coated overnight at 4° C. with 50 μl of 2μg/ml of FGFR ECD-human Fc chimeric proteins. After blocking withphosphate-buffered saline (PBS)/3% BSA, FGFR3 antibody was added andincubated at RT for 2 hours. Specifically bound FGFR3 antibody wasdetected using an HRP-conjugated anti-human Fab and the TMB peroxidasecolorigenic substrate (KPL, Gaithersburg, Md.).

To test the effect of antibodies to FGFR3 on FGF/FGFR3 interaction,FGFR3-Fc chimeric proteins were captured on Maxisorp plate coated withanti-human immunoglobulin Fcγ fragment-specific antibody (JacksonImmunoresearch, West Grove, Pa.). After wash, increasing amount of FGFR3antibody was added to the plate and incubated for 30 minutes. Then, FGF1or FGF9 and heparin were added for incubation at RT for 2 hours. Theplates were washed and incubated for 1 hour with biotinylated FGF1-specific polyclonal antibody (BAF232) or biotinylated FGF9 antibody(BAF273, R&D Systems), followed by detection with streptavidin-HRP andTMB.

Generation of Ba/F3-FGFR3 Stable Cells

cDNA encoding full-length human FGFR3 IIIb or IIIc was cloned intopQCXIP vector (Clontech Laboratories, Mountain View, Calif.) to generatepQCXIP-FGFR3-IIIb or IIIc. Specific mutations, i.e., R248C, S249C,G372C, Y375C and K652E, were introduced into the cDNA via QuickChange(Stratagene, La Jolla, Calif.). To generate Ba/F3 stable cellsexpressing wild type or mutant FGFR3, various pQCXIP-FGFR3 constructswere co-transfected into packaging cells GP2-293 with VSV-G plasmid(Clontech Laboratories). After selection with 2 μg/ml puromycin for twoweeks, cells expressing wild type or mutant FGFR3 were stained withPhycoerythrin-conjugated anti-human FGFR3 mAb (FAB766P, R&D Systems),and selected through fluorescence-activated cell sorting (FACS) forfunctional assays. For cell proliferation assay in 96-well micro-titerplate, the following cell density was used: For cells expressing wildtype FGFR3-IIIb and FGFR3-K652E: 5,000 cells/well; for the rest: 10,000cells/well. Cells were seeded in RPMI 1640 medium supplemented with 10%fetal bovine serum, 10 ng/ml FGF 1 plus 10 μg/ml heparin (Sigma-Aldrich,St. Louis, Mo.). R3Mab was added at indicated concentration and mousehybridoma FGFR3 antibodies were added at 2000 to 0.49 ng/ml (infour-fold serial dilutions) in the FGFR3-IIIb experiment and 5000 to 1.2ng/ml (in four-fold serial dilutions) in the FGFR3-IIIc experiment.After incubation for 72 hours, cell viability was assessed withCellTiter-Glo (Promega, Madison, Wis.).

Cell Proliferation Assay

For proliferation assays for RT112, RT4 and TCC-97-7 cells, 3000cells/well were seeded into 96-well micro-titer plate and were allowedto adhere overnight. The medium was then replaced with low serum medium(0.5% FBS) with control or R3Mab at concentrations indicated. Following4 days incubation, 1 μCi of [Methyl-³H] thymidine (PerkinElmer, Waltham,Mass.) was added to each well, and incubated for additional 16 hours.Cells were transferred to UniFilters using Packard Filtermate Harvester,and [³H]-thymidine incorporated into the genomic DNA of growing cellswas measured using TopCount (PerkinElmer). In some cases, cell viabilitywas assessed with CellTiter-Glo (Promega) following incubation withantibodies for 4 days. Values are presented as means+/−SE ofquadruplets.

Clonal Growth Assay

The effect of R3Mab on cell clonogenicity was assessed following apreviously described protocol (50). In brief, 400 UMUC-14 cells wereseeded into 6-well plate in DMEM medium supplemented with 10% fetalbovine serum to allow adhesion overnight. Then R3Mab or control antibodydiluted in 0.1% BSA medium was added to a final concentration of 10μg/ml. Equal volume of 0.1% BSA medium alone (Mock) was used as anothercontrol. The cells were incubated for about 12 days until cells incontrol groups formed sufficiently large colonies. Colonies were stainedwith 0.5% crystal violet, and the number and size of colonies werequantitated using GelCount (Oxford, UK). The number of colonies largerthan 120 μm in diameter was presented as mean+/−SEM (n=12).

Immunoprecipitation and Immunoblotting Analyses

To study the effect of antibodies on FGFR3 signaling, cells were starvedin serum-free medium overnight prior to the beginning of treatment.Cells were incubated with either antibodies diluted in 0.1% BSA (w/v),RPMI 1640 medium, or with 0.1% BSA medium alone (Mock). After 3 hours at37° C., FGF1 (final concentration of 15 ng/ml) and heparin (finalconcentration of 5-10 μg/ml) were added to half of the samples. Ascontrols, a similar volume of heparin alone was added to the other halfof samples. The incubation was continued for 10 min. Supernatants wereremoved by aspiration, and cells were washed with ice-cold PBS, thenlysed in RIPA buffer (Upstate, Charlottesville, Va.) supplemented with 1mM sodium orthovanadate and Complete protease inhibitor cocktail (RocheApplied Science, Indianapolis, Ind.). The lysates were cleared ofinsoluble materials by centrifugation.

FGFR3 was immunoprecipitated using a rabbit polyclonal antibody (sc-123,Santa Cruz Biotechnology, Santa Cruz, Calif.) and analyzed by sodiumdodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot.Phosphorylated FGFR3 was assessed with a monoclonal antibody againstphospho-tyrosine (4G10, Upstate). Total FGFR3 was probed with amonoclonal antibody against FGFR3 (sc-13121, Santa Cruz Biotechnology).Phosphorylation and activation of FGFR3 signaling pathway were probedusing the following antibodies: anti-FGFR^(Y653/654), anti-FRS2^(Y196),anti-phospho-p44/42 MAPK^(T2224), anti-total p44/42 MAPK andanti-AKT^(S473) were obtained from Cell Signaling Technology (Danvers,Mass.); and anti-total FRS2a (sc-8318) was purchased from Santa CruzBiotechnology (Santa Cruz, Calif.). The blots were visualized using achemiluminescent substrate (ECL Plus, Amersham Pharmacia Biotech,Piscataway, N.J.).

Antibody Epitope Mapping

To determine the epitope of R3Mab, 13 overlapping peptides, each of 15amino acids in length, were synthesized to cover the extracellulardomain of human FGFR3 from residues 138 to 310. The peptides werebiotinylated at the C-terminus, and captured on streptavidin plates(Pierce, Rockford, Ill.) overnight. After blocking with PBS/3% BSA, theplates were incubated with R3Mab and detected using an HRP-conjugatedanti-human IgG (Jackson Immunoresearch) and the TMB peroxidasecolorigenic substrate (KPL, Gaithersburg, Md.).

Mouse anti-human FGFR3 hybridoma antibodies 1G6, 6G1, and 15B2 weretested in ELISA assay to identify their binding epitopes. 1G6, 6G1 and15B2 bind to human FGFR3 IgD2-IgD3 (both IIIb and IIIc isoforms),whereas 5B8 only binds IgD2-IgD3 of human FGFR3-IIIb. In a competitionassay, 1G6, 6G1 and 15B2 competed with each other to bind human FGFR3,suggesting that 1G6, 6G1 and 15B2 have overlapping epitopes. None of thehybridoma antibodies competed with phage antibody 184.6, suggesting thatthe hybridoma antibodies have distinct epitope(s) from 184.6.

Preparation and Molecular Cloning of Mouse Anti-FGFR3 Antibodies 1G6,6G1, and 15B2

BALB/c mice were immunized 12 times with 2.0 μg of FGFR3-IIIb (rhFGFR3(IIIB)/Fc Chimera, from R&D Systems, catalog #1264-FR, lot # CYH025011,or with 2.0 μg of FGFR3-IIIc (rhFGFR3 (IIIc)/Fc Chimera, from R&DSystems, catalog #766-FR, lot # CWZ055041, resuspended in monophosphoryllipid A/trehalose dicorynomycolate adjuvant (Corixa, Hamilton, Mont.)into each hind footpad twice a week. Three days after final boost,popliteal lymph nodes were fused with mouse myeloma cell lineP3X₆₃Ag.U.1, via electrofusion (Hybrimune, Cyto Pulse Sciences, GlenBurnie, Md.). Fused hybridoma cells were selected from unfused poplitealnode or myeloma cells using hypoxanthin-aminopterin-thymidine (HAT)selection in Medium D from the ClonaCell® hybridoma selection kit(StemCell Technologies, Inc., Vancouver, BC, Canada). Culturesupernatants were initially screened for its ability to bind toFGFR3-IIIb and FGFR3-IIIc by ELISA, and hybridomas of interest weresubsequently screened for its ability to stain by FACS on transfectedFGFR3-IIIb Ba/F cells and control Ba/F, as well as antibody blockingactivity. Selected hybridomas were then cloned by limiting dilution.

Total RNA was extracted from hybridoma cells producing the mouse antihuman FGFRIII monoclonal antibody 1G6 and 15B2, using RNeasy Mini Kit(Qiagen, Germany). The variable light (VL) and variable heavy (VH)domains were amplified using RT-PCR with the following degenerateprimers:

1G6: Light chain (LC) forward: (SEQ ID NO: 195)5′-GTCAGATATCGTKCTSACMCARTCTCCWGC-3′ Heavy chain (HC) forward:(SEQ ID NO: 196) 5′-GATCGACGTACGCTGAGATCCARYTGCARCARTCTGG-3′ 6G1:Light chain (LC) forward: (SEQ ID NO: 197)5′-GTCAGATATCGTGCTGACMCARTCTCC-3′ Heavy chain (HC) forward:(SEQ ID NO: 198) 5′-GATCGACGTACGCTGAGATCCARYTGCARCARTCTGG-3′ 15B2:Light chain (LC) forward: (SEQ ID NO: 199)5′-GTACGATATCCAGATGACMCARTCTCC-3′ Heavy chain (HC) forward:(SEQ ID NO: 200) 5′-GATCGACGTACGCTGAGATCCARYTGCARCARTCTGG-3′

Light chain and Heavy chain reverse primer for all three clones are asfollowed:

Light chain reverse: (SEQ ID NO: 201) 5′-TTTDAKYTCCAGCTTGGTACC-3′Heavy chain reverse: (SEQ ID NO: 202)5′-ACAGTGGGCCCTTGGTGGAGGCTGMRGAGACDGTGASHRDRGT-3′.

The forward primers were specific for the N-terminal amino acid sequenceof the VL and VH region. The LC and HC reverse primers were designed toanneal to a region in the constant light (CL) and constant heavy domain1 (CH1), respectively, which are highly conserved across species.

Amplified VL was cloned into a pRK mammalian cell expression vector(Shields et al, (2000) J. Biol. Chem. 276:659) containing the humankappa constant domain. Amplified VH was inserted to a pRK mammalian cellexpression vector encoding the full-length human IgG1 constant domain.The sequence of the heavy and light chains was determined usingconventional methods.

Crystallization, Structure Determination and Refinement

The human FGFR3-IIIb ECD (residues 143-374) was cloned into pAcGP67Avector (BD Bioscience, San Jose, Calif.), produced in T. ni Pro cellsand purified using Ni-NTA column followed by size exclusionchromatography. The R3Mab Fab was expressed in E. coli and purifiedsequentially over a protein G affinity column, an SP sepharose columnand a Superdex 75 column. Fab-FGFR3 complex was generated by incubatingthe Fab with an excess of FGFR3 ECD, and the complex was thendeglycosylated and purified over a Superdex-200 sizing column in 20 mMTrisCl pH 7.5 and 200 mM NaCl buffer. The complex-containing fractionswere pooled and concentrated to 20 mg/ml and used in crystallizationtrials. Crystals used in the structure determination were grown at 4° C.from the following condition: 0.1 M sodium cacodylate pH 6.5, 40% MPDand 5% PEG8000 using vapor diffusion method. Data was processed usingHKL2000 and Scalepack (56). The structure was solved with molecularreplacement using program Phaser (57) and the coordinates of 1RY3(FGFR3) and 1N8Z (Fab-fragment). The model was completed using programCoot (58) and the structure refined to R/R_(free) of 20.4%/24.3% withprogram Refmac (59). Coordinates and structure factors were deposited inthe Protein Data Bank with accession code 3GRW and are also disclosed inU.S. Ser. No. 61/163,222, filed on Mar. 25, 2009, the contents of whichis hereby incorporated by reference..

ADCC Assay

Human PBMCs were isolated by Ficoll gradient centrifugation ofheparinized blood, and ADCC was measured using the multiple myeloma celllines OPM2 or KMS11 or bladder cancer cell lines RT112 or UMUC-14 astarget and PBMCs as effector cells at a 1:100 target:effector ratio. Thetarget cells (10,000 cells/well) were treated with R3Mab or with controlhuman IgG1 for 4 hours at 37° C. Cytotoxicity was determined bymeasuring LDH release using the CytoTox-ONE Homogeneous MembraneIntegrity Assay following manufacturer's instructions (Promega, Madison,Wis.). The results are expressed as percentage of specific cytolysisusing the formula: Cytotoxicity (%)=[(Experimental lysis−Experimentalspontaneous lysis)/(Target maximum lysis−target spontaneous lysis)]×100,where spontaneous lysis is the nonspecific cytolysis in the absence ofantibody, and target maximum lysis is induced by 1% Triton X-100.

Results

Inducible shRNA Knockdown of FGFR3 Attenuates Bladder Cancer Growth InVivo

As a prelude to assessing the importance of FGFR3 for bladder tumorgrowth in vivo, we examined the effect of FGFR3 knockdown in vitro.Several FGFR3 small interfering (si) RNAs effectively downregulatedFGFR3 in bladder cancer cell lines expressing either WT (RT112, RT4,SW780) or mutant (UMUC-14, S249C mutation) FGFR3. FGFR3 knockdown in allfour cell lines markedly suppressed proliferation in culture (FIGS.15A-D). Next, we generated stable RT112 cell lines expressingdoxycycline-inducible FGFR3 shRNA. Induction of three independent FGFR3shRNAs by doxycycline diminished FGFR3 expression, whereas induction ofa control shRNA targeting EGFP had no effect (FIG. 7A). In the absenceof exogenous FGF, doxycycline treatment reduced [³H]-thymidineincorporation by cells expressing different FGFR3 shRNAs, but notcontrol shRNA (FIG. 7B), confirming that FGFR3 knockdown inhibitsproliferation. Further analysis of exponentially growing RT112 cellsrevealed that FGFR3 knockdown over a 72 hr treatment with doxycyclinemarkedly and specifically reduced the percentage of cells in the S andG2 phases of the cell cycle, with a concomitant increase of cells in G1phase (FIG. 7C). Similar effect was observed with two other FGFR3 shRNAs(FIG. 16A). No significant numbers of cells with a sub-diploid DNAcontent were detected, suggesting no change in apoptosis levels. Hence,the inhibitory effect of FGFR3 knockdown on the proliferation of RT112cells is mainly due to attenuation of cell cycle progression.

We next evaluated the effect of FGFR3 knockdown on the growth ofpre-established RT112 tumor xenografts in mice. FGFR3 knockdownsubstantially and specifically suppressed tumor growth (FIG. 7D, toppanels and FIG. 16B). Analysis of day 45 tumor samples confirmedeffective FGFR3 knockdown upon doxycycline induction of FGFR3 shRNA ascompared to control shRNA (FIG. 7D, bottom panels). These resultsdemonstrate that FGFR3 is critically important both in vitro and in vivofor the growth of RT112 bladder cancer cells.

Generation of a Blocking Anti-FGFR3 Monoclonal Antibody

To examine further the importance of FGFR3 in tumor growth and toexplore the potential of this receptor as a therapeutic target, wedeveloped an antagonistic anti-FGFR3 monoclonal antibody (dubbed R3Mab)using a phage display approach. We selected this particular antibodybased on its ability to block both ligand binding and dimerization byFGFR3, and its unique capacity to inhibit not only WT FGFR3 but also themost prevalent cancer-associated mutants of this receptor (see below).R3Mab targets the extracellular IgD2 and IgD3 domains of FGFR3, whichare necessary and sufficient for FGF binding (4). R3Mab bound both theIIIb and IIIc isoforms of human FGFR3, but showed no detectable bindingto FGFR1, FGFR2 or FGFR4 (FIG. 8A). Biacore analysis indicated thatR3Mab had similar apparent affinity to murine, cynomolgus monkey andhuman FGFR3-IIIc (data not shown). The affinity of R3Mab to human FGFR3is shown in Table 2.

TABLE 2 Affinity of R3Mab to human FGFR3 determined by BIAcore analysis.R3 Mab captured on chips Human FGFR3 ECD kon/(1/Ms) koff(1/s) Kd(M) IIIb1.80E+06 2.00E−04 1.11E−10 IIIc 9.10E+04 3.20E−04 3.52E−09

We next tested the ability of R3Mab to block FGFR3 binding to FGF1 andFGF9. R3Mab strongly inhibited binding of FGF1 to FGFR3-IIIb and -IIIc,with half-maximal inhibitory concentrations (IC₅₀) of 0.3 nM and 1.7 nM,respectively (FIGS. 8B, 8C). Similarly, R3Mab efficiently blocked FGF9binding to FGFR3-IIIb and -IIIc, with an IC₅₀ of 1.1 nM and 1.3 nM,respectively (FIGS. 8D, 8E).

R3Mab Inhibits WT FGFR3 and its Most Prevalent Cancer-Associated MutantVariants

To examine whether R3Mab inhibits cell proliferation driven by WT ormutant FGFR3, we took advantage of the observation that ectopic FGFR3expression in murine pro-B cell Ba/F3 confers interleukin(IL)-3-independent, FGF1-dependent proliferation and survival (29). Inthe absence of FGF1 and IL-3, Ba/F3 cells stably expressing WT FGFR3were not viable, while FGF1 greatly enhanced their proliferation (FIG.9A). R3Mab specifically blocked FGF1-stimulated Ba/F3-FGFR3 cellproliferation in a dose-dependent manner (FIG. 9A). We next evaluatedthe impact of R3Mab on FGFR3 signaling in these cells. FGF1 inducedphosphorylation and activation of FGFR3 and concomitant activation ofp44/42 MAPK, while R3Mab effectively suppressed the activation of bothmolecules (FIG. 9B).

In bladder cancer, somatic activating mutations in FGFR3 cluster withinthe linker region between IgD2 and IgD3, the extracellular juxtamembranedomain, or the kinase domain (FIG. 9C). The extracellular missensesubstitutions most often give rise to an unpaired cysteine, leading toligand-independent dimerization of FGFR3. These mutations cause markedlydifferent levels of constitutive FGFR3 activation, possibly owing to adifferential impact on the orientation of the cytoplasmic kinase domain(30, 31). The most frequent mutations are S249C, Y375C, R248C, G372C,and K652E, which together account for 98% of all FGFR3 mutations inbladder cancer (32). We reasoned that an optimal therapeutic agentshould block not only the WT FGFR3 protein, which is overexpressed incertain cancers, but also the most prevalent tumor-associated FGFR3mutants. To assess R3Mab further, we generated Ba/F3 cell lines stablyexpressing each of the five most common FGFR3 mutant variants. Allmutants were expressed at similar levels at the cell surface, and thecysteine mutants dimerized spontaneously without ligand (data notshown). Cell lines expressing different cysteine mutants exhibited avariable growth response to FGF1, consistent with earlier findings (30,31). As previously reported (33), cells expressing FGFR3^(R248C)displayed constitutive, ligand-independent proliferation, and were notresponsive to FGF 1 (FIG. 9D). Similarly, the most frequent mutation,FGFR3^(S249C), conferred ligand-independent proliferation (FIG. 9E).Remarkably, R3Mab suppressed constitutive proliferation driven by eithermutant (FIGS. 9 D, 9E). Cells expressing the juxtamembrane domainmutations FGFR3^(G372C) (FIG. 9F) or FGFR3^(Y375C) (FIG. 9G) requiredFGF1 for proliferation, and their growth was completely blocked byR3Mab. Cells expressing FGFR3^(K652E) showed weak ligand-independentproliferation and significant growth in response to FGF1 (33). R3Mab didnot affect the weak basal activity of FGFR3^(K652E) (data not shown),but nearly abolished ligand-induced proliferation mediated by thismutant (FIG. 9H). Hence, R3Mab has a unique capacity to inhibit both WTand prevalent cancer-associated mutants of FGFR3. Moreover, R3Mab didnot display detectable agonist activity.

As a separate effort, we generated and characterized multiplemouse-anti-human FGFR3 hybridoma antibodies. None of the hybridomaantibodies could inhibit all the cancer-linked FGFR3 mutants we tested(FIG. 17), nor did they share overlapping epitopes with R3Mab.

Moreover, all of the hybridoma antibodies showed agonist activity,strongly stimulating proliferation of cancer-linked FGFR3 mutants R248Cand S249C, and showing some stimulation of proliferation of mutantsY375C and G370C. The hybridoma antibodies showed differential levels ofantagonist and agonism, depending on the FGFR3 mutant tested, asfollows:

1G6 6G1 15B2 FGFR3-IIIb wildtype inhibition inhibition inhibitionFGFR3-IIIb R248C 2X 4-5X 3-4X stimulation stimulation stimulationFGFR3-IIIbS249C 2X 4-5X 4-5X stimulation stimulation stimulationFGFR3-IIIb Y375C 1.2-1.5X 1.2-1.5X 1.2-1.5X stimulation stimulationstimulation FGFR3-IIIb K652E 50% 60-70% inhibition inhibition inhibitionFGFR3-IIIc inhibition inhibition inhibition FGFR3-IIIc G370C No effect20-30% 10-2-% inhibition inhibitionThus, the hybridoma antibodies showed unpredictable differential effecton Ba/F3 cells cell proliferation driven by various FGFR3 mutants.

Characterization of Mouse-Anti-Human FGFR3 Hybridoma Antibodies

Mouse anti-human FGFR3 hybridoma antibodies were further characterizedas follows:

(1) In an assay to test ability of anti-FGFR3 murine hybridomaantibodies to inhibit FGF1 binding to human FGFR3-IIIb and IIIcisoforms, antibodies 1G6, 6G1 and 15B2 were able to block binding ofFGF1 to human FGFR3-IIIb and IIIc isoforms in a dose-dependent manner.When tested across an antibody concentration range of about 2000 to 0.49ng/ml, antibodies 1G6, 6G1 and 15B2 blocked FGF1 binding to FGFR3-IIIbwith IC₅₀ values of 0.69, 0.87 and 0.72 nM. When tested across anantibody concentration range of about 5000 to 1.2 ng/ml, antibodies 1G6,6G1 and 15B2 blocked FGF1 binding to FGFR3-IIIc with IC₅₀ values of0.57, 3.4 and 0.7 nM, respectively.

(2) In an assay to test ability of anti-FGFR3 murine hybridomaantibodies to inhibit FGF9 binding to human FGFR3-IIIb and IIIcisoforms, antibodies 1G6, 6G1 and 15B2 efficiently blocked binding ofFGF1 to human FGFR3-IIIb and IIIc isoforms in a dose-dependent manner.When tested across an antibody concentration range of about 2000 to 0.49ng/m, antibodies 1G6, 6G1 and 15B2 blocked FGF9 binding to FGFR3-IIIbwith IC₅₀ values of 0.13, 0.16, and 0.07 nM, respectively. When testedacross an antibody concentration range of about 5000 to 1.2 ng/ml,antibodies 1G6, 6G1 and 15B2 blocked FGF9 binding to FGFR3-IIIc withIC₅₀ values of 0.13, 0.11, and 0.07 nM, respectively.

(3) The binding affinity of full-length anti-FGFR3 murine hybridomaantibodies 1G6, 6G1 and 15B2 was determined using Biacore analysis. Theresults of this analysis are shown in Table 3.

TABLE 3 FGFR3-IIIB FGFR3-IIIC kon kon (10⁵ koff Kd (10⁵ koff Kd AntibodyM⁻¹s⁻¹) (10⁻⁴s⁻¹) (nM) M⁻¹s⁻¹) (10⁻⁴s⁻¹) (nM) 1G6 mIgG 2.2 3.1 1.4 2.22.8 1.3 6G1 mIgG 2.7 3.8 1.4 2.6 3.2 1.2 15B2 mIgG 4.1 29 7.1 3.5 3911.1

(4) In an assay to test ability of anti-FGFR3 murine hybridomaantibodies to inhibit Ba/F3 cell proliferation driven by humanFGFR3-IIIb or IIIc, antibodies 1G6, 6G1 and 15B2 were able to blockBa/F3 cell proliferation driven by human FGFR3-IIIb or IIIc in adose-dependent manner. When tested across an antibody concentrationrange of about 0.01 to 100 ug/ml, antibodies 1G6, 6G1 and 15B2 blockedBa/F3 cell proliferation driven by FGFR3-IIIb with IC₅₀ values of 3-5nM, 3 nM, and 6-8 nM, respectively, and blocked Ba/F3 cell proliferationdriven by FGFR3-IIIc with IC₅₀ values of 10-35 nM, 24 nM, and 60 nM,respectively.

(5) In an assay to test ability of anti-FGFR3 murine hybridomaantibodies to inhibit FGF1-induced signaling in Ba/F3 cells expressinghuman FGFR3-IIIb, antibodies 1G6, 6G1 and 15B2 were able to block FGF1-induced signaling in Ba/F3 cells expressing human FGFR3-IIIb in adose-dependent manner when tested across an antibody concentration rangeof about 0.25 to 6.75 ug/ml. 25 ng/ml of FGF1 was used in thisexperiment. In the absence of FGF1, antibody treatment had no effect onFGFR3 activation.

(6) In an assay to test ability of anti-FGFR3 murine hybridomaantibodies to inhibit FGF1-induced signaling in Ba/F3 cells expressinghuman FGFR3-IIIc, antibodies 1G6, 6G1 and 15B2 were able to block FGF1-induced signaling in Ba/F3 cells expressing human FGFR3-IIIc in adose-dependent manner when tested across an antibody concentration rangeof about 0.25 to 6.75 ug/ml. 25 ng/ml of FGF1 was used in thisexperiment. In the absence of FGF1, antibody treatment had no effect onFGFR3 activation.

Structural Basis for the Interaction of R3Mab with FGFR3

To gain insight into R3Mab's mode of interaction with FGFR3, wesynthesized a panel of 13 overlapping peptides spanning the FGFR3-IIIbIgD2 and D3 regions and tested their binding to R3Mab. Peptides 3(residues 164-178) and 11 (residues 269-283) showed specific binding toR3Mab, with peptide 3 having a stronger interaction (FIG. 10A),indicating that the corresponding regions on FGFR3 are critical forrecognition by R3Mab. Previous crystallographic studies of FGFR1 incomplex with FGF2 identified critical receptor residues engaged indirect binding to FGF and heparin as well as in receptor dimerization(34). Alignment of FGFR3 peptides 3 and 11 with the functionallyimportant sites in FGFR1 revealed that these peptides encompasscorresponding FGFR1 residues essential for direct FGF2 binding, receptordimerization, as well as interaction with heparin (FIG. 10B). These dataindicate that the epitope of R3Mab on FGFR3 overlaps with receptorresidues engaged in ligand association and receptor-receptorinteraction.

We next crystallized the complex between the Fab fragment of R3Mab andthe extracellular IgD2-D3 region of human FGFR3-IIIb, and determined theX-ray structure at 2.1 Å resolution (FIGS. 10 C, 10D; Table 4). In thiscomplex, approximately 1400 Å2 and 1500 Å2 of solvent-accessible surfaceareas are buried on FGFR3 and the Fab, respectively. About 80% of theburied interface involves IgD2, while the remainder entails the linkerand IgD3 regions. On the Fab side of the complex, about 40% of theburied interface involve complementarity-determining region (CDR)-H3,20% CDR-H2, 20% CDR-L2, and minor contributions are from other CDRs andframework residues. Notably, amino acids (AAs) from CDR-H3 form twoβ-strands, which extend the β-sheet of IgD2 (FIG. 10D). The Fabinteracts with AAs that constitute the FGF binding site of FGFR3 as wellas residues that form the receptor dimerization interface, as previouslyidentified in various dimeric FGF:FGFR complexes (e.g., PDB code 1CVS,(34); and FIG. 10C, areas in grey/crosshatched and dark grey). Theinteraction interfaces identified by crystallography were fullyconsistent with the peptide-based data (FIGS. 18 A, 18B). Together,these results reveal how R3Mab inhibits ligand binding, and furthersuggest that binding of R3Mab to FGFR3 may prevent receptordimerization. FGFR3 amino acids that contact R3Mab are shown in Table 5.Crystallographic coordinates for this structure are deposited in theProtein Data Bank with accession code 3GRW.

TABLE 4 Summary of crystallographic analysis Data collection FGFR3-IIIb:R3Mab Fab Space group P2₁2₁2₁ Cell parameters a = 58.5, b = 99.3, c =143.7 Resolution (Å) 25-2.1 R_(sym) ^(a) 0.098 (0.663)^(b) Number ofobservations 288498 Unique reflections 49851 Completeness (%)  99.9(100.0)^(b) Refinement Resolution (Å) 20-2.1 Number of reflections 46714Final R^(c), R_(free) (F > 0) 0.187, 0.224 Number of non-H atoms 5270Number of Amino Acids 650 Sulfate 1 Sugar 1 Solvent atoms 274 Rmsd bonds(Å) 0.011 Rmsd angles (°) 1.4 ^(a)R_(sym) = Σ|I − <I>|/Σ I. <I> is theaverage intensity of symmetry related observations of a uniquereflection. ^(b)Numbers in parentheses refer to the highest resolutionshell. ^(c)R = Σ|F_(o) − F_(c)|/ΣF_(o). R_(free) is calculated as R, butfor 5% of the reflections excluded from all refinement.

TABLE 5 Residues in FGFR3 that are in contact with R3Mab Residue Buriedsurface of residue in the interface THR 154 0.10 ARG 155 16.50 ARG 158105.40 MET 159 6.00 LYS 161 52.50 LYS 162 1.70 LEU 163 12.30 LEU 16455.10 ALA 165 10.10 VAL 166 10.60 PRO 167 45.50 ALA 168 22.60 ALA 16963.60 ASN 170 75.40 THR 171 83.00 VAL 172 1.70 ARG 173 91.70 PHE 1741.50 ARG 175 95.60 PRO 177 15.90 GLY 202 2.10 LYS 205 63.40 ARG 20767.60 GLN 210 31.60 SER 212 0.40 VAL 214 26.40 GLU 216 48.90 SER 2171.80 TYR 241 15.90 LEU 246 3.10 GLU 247 1.80 ARG 248 46.90 TYR 278 32.20SER 279 1.80 ASP 280 19.80 ALA 281 3.00 GLN 282 24.80 PRO 283 0.50 SER314 1.20 GLU 315 82.60 SER 316 33.20 VAL 317 56.60 GLU 318 51.50

We compared the R3Mab-FGFR3 structure with a previously publishedstructure of FGFR3-IIIc in complex with FGF1 (4, 35) (FIGS. 10E, 10F).Superposition of the antibody-receptor and ligand-receptor complexesrevealed that there are no major conformational differences within theindividual receptor domains, except in the region that distinguishesFGFR3-IIIc from FGFR3-IIIb; however, the orientation of IgD3 relative toIgD2 was drastically different (FIG. 10E, white and grey; FIG. 10F,white and grey-mesh). Since the relative positions of IgD2 and IgD3 arecritical for ligand binding, the alternate conformation adopted by IgD3upon R3Mab binding may provide an additional mechanism to prevent ligandinteraction with FGFR3.

R3Mab Inhibits Endogenous WT and Mutant FGFR3 in Bladder Cancer Cells

To assess whether R3Mab could suppress FGFR3 function in bladder cancercells, we first examined RT112 and RT4 cell lines, which express WTFGFR3. R3Mab strongly inhibited [³H]-thymidine incorporation by RT112cells (FIG. 11A) and exerted a significant, though more moderatesuppression of RT4 cell proliferation (FIG. 19A). To investigate R3Mab'seffect on FGFR3 activation, we examined the phosphorylation of FGFR3 inRT112 cells. Consistent with the results in Ba/F3-FGFR3 cells (FIG. 9B),R3Mab markedly attenuated FGF1-induced FGFR3 phosphorylation (FIG. 11B).We next examined phosphorylation of FRS2a, AKT, and p44/42 MAPK, threedownstream mediators of FGFR3 signaling. FGF 1 strongly activated thesemolecules in RT112 cells, while R3Mab significantly diminished thisactivation (FIG. 11B). Similarly, R3Mab suppressed FGF1-inducedphosphorylation of FGFR3 and MAPK in RT4 cells (FIG. 19B).

We next investigated whether R3Mab could inhibit activation ofendogenous mutant FGFR3 in human bladder cancer cells. S249C is the mostfrequent FGFR3 mutation in bladder cancer (FIG. 9C). Two available celllines, UMUC-14 and TCC-97-7, carry a mutated FGFR3^(S249C) allele (Ref.36 and data not shown). Although R3Mab did not affect the exponentialgrowth of UMUC-14 cells in culture (data not shown), it significantlyreduced the clonal growth of these cells (FIG. 11C). Specifically, R3Mabdecreased the number of colonies larger than 120 μm in diameterapproximately by 77% as compared with control antibody (FIG. 11D).Furthermore, R3Mab inhibited [³H]-thymidine incorporation by TCC-97-7cells in culture (FIG. 19C).

The S249C mutation is reported to result in ligand-independentactivation of FGFR3 (26, 30). Indeed, FGFR3^(S249C) was constitutivelyphosphorylated irrespective of FGF1 treatment in UMUC-14 cells andTCC-97-7 cells, while R3Mab reduced constitutive phosphorylation ofFGFR3^(S249C) as compared with control antibody in both cell lines(FIGS. 11E, 19D).

R3Mab Inhibits Dimer Formation by FGFR3^(S249C)

The ability of R3Mab to inhibit constitutive FGFR3^(S249C) signaling andproliferation in bladder cancer cells was surprising, considering thatthis mutant can undergo disulfide-linked, ligand-independentdimerization (26, 30). To explore how R3Mab inhibits FGFR3^(S249C), weexamined the effect of R3Mab on the oligomeric state of this mutant inUMUC-14 cells. Under reducing conditions, FGFR3^(S249C) migrated as asingle band of −97 kDa, consistent with monomeric size (FIG. 12A). Undernon-reducing conditions, in cells treated with control antibody a largefraction of FGFR3^(S249C) appeared as a band of −200 kDa, regardless ofFGF1 addition, indicating a constitutive dimeric state (FIG. 12A). R3Mabtreatment substantially decreased the amount of dimers, with aconcomitant increase in monomers (FIG. 12A). Consistently, R3Mabdecreased the level of FGFR3^(S249C) dimers in TCC-97-7 cellsirrespective of FGF1 treatment (FIG. 19E).

How does R3Mab decrease the FGFR3^(S249C) dimer levels in bladder cancercells? One potential explanation is that it may disrupt theFGFR3^(S249C) dimer through antibody-induced FGFR3 internalization andtrafficking through endosomes or lysosomes. We tested this possibilityby pharmacologically intervening with endocytosis. R3Mab nonethelessdecreased the amount of dimer in UMUC-14 cells pre-treated with variousendocytosis inhibitors, despite substantial blockade of FGFR3^(S249C)internalization (FIG. 20 A, 20B). Thus, dimer disruption by R3Mab isindependent of endocytosis. Another possible explanation is thatcellular FGFR3^(S249C) may exist in a dynamic monomer-dimer equilibrium;accordingly, binding of R3Mab to monomeric FGFR3^(S249C) could preventdimer formation and thereby shift the equilibrium toward the monomericstate. To examine this possibility, we used the non-cell-permeatingagent 5,5′Dithiobis 2-nitrobenzoic acid (DTNB), which selectively reactswith and blocks free sulfhydryl groups of unpaired cysteines (37).Treatment of UMUC-14 cells with DTNB led to the accumulation ofFGFR3^(S249C) monomers at the expense of dimers (FIG. 12B), indicatingthat FGFR3^(S249C) exists in a dynamic equilibrium between monomers anddimers.

To test whether R3Mab affects this equilibrium, we generated a solublerecombinant protein comprising the IgD2-D3 domains of FGFR3^(S249C) andisolated the dimers by size exclusion chromatography. We incubated thedimers with buffer or antibodies in the presence of a very lowconcentration of reducing agent (25 μM of DTT), and analyzed theoligomeric state of the receptor by SDS-PAGE under non-reducingconditions. R3Mab significantly accelerated the appearance of a ˜25 kDaband representing monomeric FGFR3^(S249C) at the expense of the ˜50 kDadimer, as compared with mock or antibody controls (FIG. 12C); indeed, by2 hr the decrease in dimers was substantially more complete in thepresence of R3Mab. These results indicate that R3Mab shifts theequilibrium between the monomeric and dimeric states of FGFR3^(S249C) infavor of the monomer.

R3Mab does not Promote FGFR3 Down-Regulation

We examined the effect of R3Mab (clone 184.6.1) and anti-FGFR3 hybridomaantibodies on FGFR3 downregulation by analyzing FGFR3 internalizationand degradation in FGFR3 antibody-treted cells. Bladder cancer celllines expressing wild type FGFR3 (RT112) or mutated FGFR3 (S249C inTCC97-7) were treated with R3Mab or hybridoma antibodies 1G6 or 6G1 for4 to 24 hours, then cell lysates were harvested for western blotanalysis of total FGFR3 levels. Treatment with R3 Mab did not reduceFGFR3 levels, while treatment with hybridoma mabs 1G6 and 6G1significantly reduced FGFR3 levels. These results suggested that R3Mabdid not promote FGFR3 down-regulation while mabs 1G6 and 6G1 did promoteFGFR3 receptor internalization and down regulation. In a separateexperiment, surface FGFR3 levels were examined using FACS analysis.After 24 hours of R3Mab (clone 184.6.1) treatment of UMUC-14 cells(containing FGFR3 S249C mutation), cell surface FGFR3 levels slightlyincreased. These results demonstrate that R3Mab treatment did notpromote FGFR3 down-regulation.

R3Mab Inhibits Growth and FGFR3 Signaling in Multiple Tumor Models

Next, we examined the effect of R3Mab on the growth of bladder cancercells in vivo. We injected nu/nu mice with RT112 cells (which express WTFGFR3), allowed tumors to grow to a mean volume of ˜150 mm³, and dosedthe animals twice weekly with vehicle or R3Mab. Compared with vehiclecontrol at day 27, R3Mab treatment at 5 or 50 mg/kg suppressed tumorgrowth by about 41% or 73% respectively (FIG. 13A). Analysis of tumorlysates collected 48 hr or 72 hr after treatment showed that R3Mabmarkedly decreased the level of phosphorylated FRS2a (FIG. 13B).Intriguingly, total FRS2a protein levels were also lower inR3Mab-treated tumors, suggesting that FGFR3 inhibition may further leadto downregulation of FRS2a. R3Mab also lowered the amount ofphosphorylated MAPK in tumors, without affecting total MAPK levels (FIG.13B). Thus, R3Mab inhibits growth of RT112 tumor xenografts inconjunction with blocking signaling by WT FGFR3.

We next investigated the effect of R3Mab on growth of xenograftsexpressing mutant FGFR3. R3Mab treatment profoundly attenuated theprogression of Ba/F3-FGFR3^(S249C) tumors (FIG. 13C). Moreover, R3Mabsignificantly inhibited growth of UMUC-14 bladder carcinoma xenografts(FIG. 13D). To evaluate whether R3Mab impacts FGFR3^(S249C) activationin vivo, we assessed the level of FGFR3^(S249C) dimer in tumor lysatescollected 24 hr or 72 hr after treatment. Under non-reducing conditions,the amount of FGFR3^(S249C) dimer was substantially lower in R3Mabtreated tumors as compared with control group, whereas totalFGFR3^(S249C) levels, as judged by the amount detected under reducingconditions, showed little change (FIG. 13E). No apparent accumulation ofFGFR3^(S249C) monomer was observed in tumor lysates, in contrast to theresults in cell culture (FIG. 13E vs. 12A). This could be due to theweak detection sensitivity for monomeric FGFR3 under non-reducingconditions by the rabbit polyclonal anti-FGFR3 antibody used in thisstudy (FIG. 21). Importantly, R3Mab also significantly inhibited thephosphorylation and activation of MAPK in UMUC-14 tumors (FIG. 13E),suggesting that R3Mab inhibits the activity of FGFR3^(S249C) in vivo. Wedid not observe any significant weight loss or other gross abnormalitiesin any the in vivo studies. Furthermore, in a safety study conducted inmice, R3Mab, which binds with similar affinity to both human and murineFGFR3, did not exert any discernable toxicity in any organs, includingbladder (data not shown). Together, these data indicate that multipleexposures to R3Mab are well tolerated in mouse.

Anti-Tumor Activity of R3Mab in Multiple Myeloma Xenograft ModelsInvolves ADCC

To assess whether R3Mab might harbor therapeutic potential for multiplemyeloma, we first tested the effect of R3Mab on the proliferation andsurvival of three t(4; 14)+ cell lines in culture. UTMC-2 cells carry WTFGFR3, while OPM2 and KMS11 harbor a K650E and Y373C substitution,respectively (7). In culture, R3Mab abrogated FGF9-induced proliferationof UTMC-2 cells completely (FIG. 22A). R3Mab modestly inhibited thegrowth of OPM2 cells, but had no apparent effect on the proliferation ofKMS11 cells (FIGS. 22 B, 22C). Since UTMC-2 cells do not form tumors inmice, we evaluated the efficacy of R3Mab against OPM2 and KMS11 tumors.R3Mab almost completely abolished xenograft tumor growth of both celllines (FIGS. 14 A, 14B).

The marked difference in activity of R3Mab against OPM2 and KMS11 tumorcells in vitro and in vivo suggested the possibility that R3Mab may becapable of supporting Fc-mediated immune effector functions againstthese FGFR3-overexpressing tumors. Both cell lines express high levelsof CD55 and CD59 (data not shown), two inhibitors of the complementpathway; accordingly, no complement-dependent cytotoxicity was observed(data not shown). We then focused on ADCC. ADCC occurs when an antibodybinds to its antigen on a target cell, and via its Fc region, engagesFcγ receptors (FcγRs) expressed on immune effector cells (38). To testADCC in vitro, we incubated KMS11 or OPM2 cells with freshly isolatedhuman peripheral blood mononuclear cells (PBMC) in the presence of R3Mabor control antibody. R3Mab mediated significant PBMC cytolytic activityagainst both myeloma cell lines (FIGS. 14 C, 14D). By contrast, R3Mabdid not support cytolysis of bladder cancer RT112 or UMUC-14 cells(FIGS. 14 E, 14F). As measured by Scatchard analysis, the multiplemyeloma cells express substantially more cell-surface FGFR3 than thebladder carcinoma cell lines (˜5-6 fold more receptors per cell; FIGS.23 A, 23B).

To address the contribution of ADCC to the activity of R3Mab in vivo, weintroduced the previously characterized D265A/N297A (DANA) mutation intothe antibody's Fc domain. This dual substitution in the Fc domain of anantibody abolishes its binding to FcγRs (39), preventing recruitment ofimmune effector cells. The DANA mutation did not alter R3Mab binding toFGFR3 or inhibition of FGFR3 activity in vitro, nor did it change thepharmacokinetics of R3Mab in mice (data not shown); however, itsubstantially abolished in vivo activity against OPM2 or KMS11xenografts (FIGS. 14 G, 14H). By contrast, the DANA mutation did notalter the anti-tumor activity of R3Mab towards RT112 and UMUC-14 bladdercancer xenografts (FIGS. 24 A, 24B). Together, these results suggestthat Fc-dependent ADCC plays an important role in the efficacy of R3Mabagainst OPM2 and KMS11 multiple myeloma xenografts.

Additional Xenograft Studies

R3Mab (clone 184.6.1N54S) was further characterized as follows:

-   -   (a) R3Mab was tested for in vivo efficacy using a tumor        xenograft model based on a liver cancer cell line (Huh7)        essentially as described above. When tested at an antibody        concentration of 5 mg/kg and 30 mg/kg, R3Mab significantly        inhibited tumor growth in vivo. Tumor growth was inhibited about        50% compared to tumor growth in control animals.    -   (b) R3Mab was tested for in vivo efficacy using a tumor        xenograft model based on a breast cancer cell line (Cal-51)        which expressed FGFR3 essentially as described above. Results        from this efficacy study showed that the R3Mab antibody was        capable of inhibiting tumors in vivo when tested at antibody        concentration range of about 1 mg/kg to 100 mg/kgs. Tumor growth        was inhibited about 30% compared to tumor growth in control        animals.

DISCUSSION

The association of FGFR3 overexpression with poor prognosis in t(4; 14)+multiple myeloma patients and the transforming activity of activatedFGFR3 in several experimental models have established FGFR3 as animportant oncogenic driver and hence a potential therapeutic target inthis hematologic malignancy. By contrast, despite reports of a highfrequency of mutation and/or overexpression of FGFR3 in bladdercarcinoma (24, 25, 40), a critical role for FGFR3 signaling in thisepithelial malignancy has not been established in vivo. Moreover, thetherapeutic potential of FGFR3 inhibition in bladder cancer has yet tobe defined. Here we show that genetic or pharmacological interventionwith FGFR3 inhibits growth of several human bladder cancer xenografts inmice. These results demonstrate that FGFR3 function is critical fortumor growth in this setting, underscoring the potential importance ofthis receptor as an oncogenic driver and therapeutic target in bladdercancer. Blockade of FGFR3 function inhibited growth of xenograftsexpressing either WT or mutant FGFR3 alike, suggesting that both formsof the receptor may contribute significantly to bladder tumorprogression. Albeit much less frequently than in bladder cancer, FGFR3mutations or overexpression have been identified in other solid tumormalignancies, including cervical carcinoma (40), hepatocellularcarcinoma (41) and non-small cell lung cancer (42, 43), suggesting apotential contribution of FGFR3 to additional types of epithelialcancer.

The apparent involvement of FGFR3 in diverse malignancies identifiesthis receptor as an intriguing candidate for targeted therapy. Whilesmall molecule compounds that can inhibit FGFR3 kinase activity havebeen described (18-22, 44), the close homology of the kinase domainswithin the FGFR family has hampered the development of FGFR3-selectiveinhibitors. The lack of selectivity of the reported inhibitors makes itdifficult to discern the relative contribution of FGFR3 to the biologyof specific cancer types; further, it may carry safety liabilities,capping maximal dose levels and thus limiting optimal inhibition ofFGFR3. Therefore, to achieve selective and specific targeting of FGFR3,we turned to an antibody-based strategy. We reasoned that an optimaltherapeutic antibody should be capable of blocking not only the WT butalso the prevailing cancer-linked mutants of FGFR3. Furthermore, giventhat dimerization of FGFR3 is critical for its activation, an antibodythat not only blocks ligand binding but also interferes with receptordimerization could be superior. Additional desirable properties wouldinclude the ability to support Fc-mediated effector function and thelong serum half-life conferred by the natural framework of a full-lengthantibody. We focused our screening and engineering efforts to identifyan antibody molecule that combines all of these features, leading to thegeneration of R3Mab. Binding studies demonstrated the ability of R3Mabto compete with FGF ligands for interaction with both the IIIb and IIIcisoforms of FGFR3. Further experiments with transfected BaF/3 cell linesconfirmed the remarkable ability of R3Mab to block both WT and prevalentcancer-associated FGFR3 mutants. In addition, R3Mab exerted significantanti-tumor activity in several xenograft models of bladder cancerexpressing either WT FGFR3 or FGFR3^(S249C) which is the most commonmutant of the receptor in this disease. Pharmacodynamic studiessuggested that the anti-tumor activity R3Mab in these models is based oninhibition of FGFR3 signaling, evident by diminished phosphorylation ofits downstream mediators FRS2a and MAPK. These data further reinforcethe conclusion that FGFR3 is required for bladder tumor progression, asdemonstrated by our FGFR3 shRNA studies.

FGFR3 mutations in bladder cancer represent one of the most frequentoncogenic alterations of a protein kinase in solid tumor malignancies,reminiscent of the common mutation of B-Raf in melanoma (45). Most ofthe activating mutations in FGFR3 give rise to an unpaired cysteine,leading to ligand-independent receptor dimerization and to variousdegrees of constitutive activation. A previous study using a monovalentanti-FGFR3 Fab fragment indicated differential inhibitory activityagainst specific FGFR3 mutants (46); however, the molecular basis forthis variable effect was not investigated. Compared with monovalentantibody fragments, bivalent antibodies have the capacity to induce theclustering of antigens, and in the case of receptor tyrosine kinases,may cause receptor oligomerization and activation. Despite itsfull-length, bivalent configuration, R3Mab displayed universalinhibition of WT FGFR3 and of a wide spectrum of FGFR3 mutants,including variants that are ligand-dependent (FGFR3^(G372C),FGFR3^(Y375C)), constitutively active (FGFR3^(R248)C, FGFR3^(S249C)), orboth (FGFR3^(K652E)). These results raise the question: How does R3Mabantagonize both WT and various FGFR3 mutants, including disulfide-linkedvariants?

Based on sequence alignment with FGFR1, the peptide epitope recognizedby R3Mab overlaps with FGFR3 residues involved in binding to ligand andheparin, as well as receptor dimerization. This conclusion was confirmedby crystallographic studies of the complex between R3Mab and theextracellular regions of FGFR3. The X-ray structure revealed that theantibody binds to regions of IgD2 and IgD3 that are critical forligand-receptor interaction as well as receptor-receptor contact. Thus,R3Mab may block WT FGFR3 both by competing for ligand binding and bypreventing receptor dimerization. R3Mab may employ a similar mechanismto inhibit FGFR3^(K652E)which has low constitutive activity, butrequires ligand for full activation. Furthermore, R3Mab binding changesthe relative orientation of FGFR3 IgD3 with respect to IgD2. Thisfinding raises the formal possibility that the antibody might alsoinhibit receptor activation by forcing a conformation that is notconducive to signal transduction—a notion that requires further study.

To gain better insight into how R3Mab blocks FGFR3 variants possessingan unpaired cysteine, we analyzed the most common mutant, FGFR3^(S249C),in greater detail. Experiments with the free-sulfhydryl blocker DTNBindicated a dynamic equilibrium between the monomeric and dimeric stateof FGFR3^(S249C). Similar equilibrium between oxidized and reducedstates modulated by endogenous redox regulators has been reported forNMDA receptors (46). Incubation of bladder cancer cells expressingFGFR3^(S249C) with R3Mab led to a decline in the amount of receptordimers and a concomitant increase in the level of monomers. Moreover,the purified IgD2-D3 fragment of FGFR3^(S249C) formed dimers insolution; when incubated with R3Mab, the dimers steadily disappearedwhile monomeric FGFR3^(S249C) accumulated. Taken together with thestructural analysis, these results suggest that R3Mab captures monomericFGFR3^(S249C) and hinders its dimerization. Over time, R3Mab shifts theequilibrium towards the monomeric state, blocking constitutive receptoractivity. This mechanism might also explain how R3Mab inhibits othercysteine mutants of FGFR3.

Another important finding of this study was the potent anti-tumoractivity of R3Mab against the t(4;14)+ multiple myeloma cell lines OPM2and KMS11 in vivo. By contrast, R3Mab had modest to minimal impact onproliferation or survival of these cells in culture. OPM2 and KMS11cells express relatively high cell surface levels of FGFR3 (5-6 foldhigher than RT112 and UMUC-14 bladder carcinoma cells). These higherantigen densities may permit R3Mab to support efficient recruitment ofFcγR-bearing immune effector cells and activation of ADCC. Indeed, inthe presence of human PBMC, R3Mab mediated cytolysis of OPM2 and KMS11cells, but not RT112 or UMUC-14 bladder cancer cells. Moreover, the DANAmutant version of R3Mab, which is incapable of FcγR binding, had noeffect on KMS11 or OPM2 growth in vivo, but still suppressed growth ofRT112 and UMUC-14 tumors similarly to R3Mab. Together, these dataindicate that R3Mab has a dual mechanism of anti-tumor activity: (a) Incells expressing lower surface levels of WT or mutant FGFR3, it blocksligand-dependent or constitutive signaling; (b) In cells expressingrelatively high surface FGFR3 levels, it induces ADCC.

Our results also raise some new questions. First, it is unknown why thebladder cancer cell lines tested in this study display variablesensitivity to R3Mab. Such differential response, which is common fortargeted therapy, may be a reflection of the distinct genetic make-up ofindividual tumors. Indeed, Her2-positive breast cancer cells showvariable sensitivity to anti-Her2 antibody (48), as do various cancercells in response to anti-EGFR antibody (49). In this context,development of additional in vivo models for bladder cancer with WT andmutant FGFR3 is urgently needed to assess sensitivity to FGFR3 moleculesin animals. Moreover, elucidation of predictive biomarkers may helpidentify patients who can optimally benefit from FGFR3-targeted therapy.Secondly, because R3Mab did not induce tumor regression in the models weexamined, future studies should explore whether R3Mab can cooperate withestablished therapeutic agents.

In conclusion, our findings implicate both WT and mutant FGFR3 asimportant for bladder cancer growth, thus expanding the in vivooncogenic involvement of this receptor from hematologic to epithelialmalignancy. Furthermore, our results demonstrate that both WT and mutantFGFR3 can be effectively targeted in tumors with a full-length antibodythat combines the ability to block ligand binding, receptor dimerizationand signaling, as well as to promote tumor cell lysis by ADCC. Theseresults provide a strong rationale for investigating antibody-based,FGFR3-targeted therapies in diverse malignancies associated with thisreceptor.

PARTIAL REFERENCE LIST

-   1. Eswarakumar, V. P., Lax, I., and Schlessinger, J. 2005. Cellular    signaling by fibroblast growth factor receptors. Cytokine Growth    Factor Rev 16:139-149.-   2. L'Hote, C. G., and Knowles, M. A. 2005. Cell responses to FGFR3    signalling: growth, differentiation and apoptosis. Exp Cell Res    304:417-431.-   3. Dailey, L., Ambrosetti, D., Mansukhani, A., and    Basilico, C. 2005. Mechanisms underlying differential responses to    FGF signaling. Cytokine Growth Factor Rev 16:233-247.-   4. Mohammadi, M., Olsen, S. K., and Ibrahimi, O. A. 2005. Structural    basis for fibroblast growth factor receptor activation. Cytokine    Growth Factor Rev 16:107-137.-   5. Grose, R., and Dickson, C. 2005. Fibroblast growth factor    signaling in tumorigenesis. Cytokine Growth Factor Rev 16:179-186.-   6. Chang, H., Stewart, A. K., Qi, X. Y., Li, Z. H., Yi, Q. L., and    Trudel, S. 2005. Immunohistochemistry accurately predicts FGFR3    aberrant expression and t(4; 14) in multiple myeloma. Blood    106:353-355.-   7. Chesi, M., Nardini, E., Brents, L. A., Schrock, E., Ried, T.,    Kuehl, W. M., and Bergsagel, P. L. 1997. Frequent translocation    t(4;14)(p16.3;q32.3) in multiple myeloma is associated with    increased expression and activating mutations of fibroblast growth    factor receptor 3. Nat Genet 16:260-264.-   8. Fonseca, R., Blood, E., Rue, M., Harrington, D., Oken, M. M.,    Kyle, R. A., Dewald, G. W., Van Ness, B., Van Wier, S. A.,    Henderson, K. J., et al. 2003. Clinical and biologic implications of    recurrent genomic aberrations in myeloma. Blood 101:4569-4575.-   9. Moreau, P., Facon, T., Leleu, X., Morineau, N., Huyghe, P.,    Harousseau, J. L., Bataille, R., and Avet-Loiseau, H. 2002.    Recurrent 14q32 translocations determine the prognosis of multiple    myeloma, especially in patients receiving intensive chemotherapy.    Blood 100:1579-1583.-   10. Pollett, J. B., Trudel, S., Stern, D., Li, Z. H., and    Stewart, A. K. 2002. Overexpression of the myeloma-associated    oncogene fibroblast growth factor receptor 3 confers dexamethasone    resistance. Blood 100:3819-3821.-   11. Bernard-Pierrot, I., Brams, A., Dunois-Larde, C., Caillault, A.,    Diez de Medina, S. G., Cappellen, D., Graff, G., Thiery, J. P.,    Chopin, D., Ricol, D., et al. 2006. Oncogenic properties of the    mutated forms of fibroblast growth factor receptor 3b.    Carcinogenesis 27:740-747.-   12. Agazie, Y. M., Movilla, N., Ischenko, I., and    Hayman, M. J. 2003. The phosphotyrosine phosphatase SHP2 is a    critical mediator of transformation induced by the oncogenic    fibroblast growth factor receptor 3. Oncogene 22:6909-6918.-   13. Ronchetti, D., Greco, A., Compasso, S., Colombo, G., Dell'Era,    P., Otsuki, T., Lombardi, L., and Neri, A. 2001. Deregulated FGFR3    mutants in multiple myeloma cell lines with t(4;14): comparative    analysis of Y373C, K650E and the novel G384D mutations. Oncogene    20:3553-3562.-   14. Chesi, M., Brents, L. A., Ely, S. A., Bais, C., Robbiani, D. F.,    Mesri, E. A., Kuehl, W. M., and Bergsagel, P. L. 2001. Activated    fibroblast growth factor receptor 3 is an oncogene that contributes    to tumor progression in multiple myeloma. Blood 97:729-736.-   15. Plowright, E. E., Li, Z., Bergsagel, P. L., Chesi, M.,    Barber, D. L., Branch, D. R., Hawley, R. G., and    Stewart, A. K. 2000. Ectopic expression of fibroblast growth factor    receptor 3 promotes myeloma cell proliferation and prevents    apoptosis. Blood 95:992-998.-   16. Chen, J., Williams, I. R., Lee, B. H., Duclos, N., Huntly, B.    J., Donoghue, D. J., and Gilliland, D. G. 2005. Constitutively    activated FGFR3 mutants signal through PLCgamma-dependent and    -independent pathways for hematopoietic transformation. Blood    106:328-337.-   17. Li, Z., Zhu, Y. X., Plowright, E. E., Bergsagel, P. L., Chesi,    M., Patterson, B., Hawley, T. S., Hawley, R. G., and    Stewart, A. K. 2001. The myeloma-associated oncogene fibroblast    growth factor receptor 3 is transforming in hematopoietic cells.    Blood 97:2413-2419.-   18. Trudel, S., Ely, S., Farooqi, Y., Affer, M., Robbiani, D. F.,    Chesi, M., and Bergsagel, P. L. 2004. Inhibition of fibroblast    growth factor receptor 3 induces differentiation and apoptosis in    t(4;14) myeloma. Blood 103:3521-3528.-   19. Trudel, S., Li, Z. H., Wei, E., Wiesmann, M., Chang, H., Chen,    C., Reece, D., Heise, C., and Stewart, A. K. 2005. CHIR-258, a    novel, multitargeted tyrosine kinase inhibitor for the potential    treatment of t(4;14) multiple myeloma. Blood 105:2941-2948.-   20. Chen, J., Lee, B. H., Williams, I. R., Kutok, J. L.,    Mitsiades, C. S., Duclos, N., Cohen, S., Adelsperger, J., Okabe, R.,    Coburn, A., et al. 2005. FGFR3 as a therapeutic target of the small    molecule inhibitor PKC412 in hematopoietic malignancies. Oncogene    24:8259-8267.-   21. Paterson, J. L., Li, Z., Wen, X. Y., Masih-Khan, E., Chang, H.,    Pollett, J. B., Trudel, S., and Stewart, A. K. 2004. Preclinical    studies of fibroblast growth factor receptor 3 as a therapeutic    target in multiple myeloma. Br J Haematol 124:595-603.-   22. Grand, E. K., Chase, A. J., Heath, C., Rahemtulla, A., and    Cross, N. C. 2004. Targeting FGFR3 in multiple myeloma: inhibition    of t(4;14)-positive cells by SU5402 and PD173074. Leukemia    18:962-966.-   23. Gomez-Roman, J. J., Saenz, P., Molina, M., Cuevas Gonzalez, J.,    Escuredo, K., Santa Cruz, S., Junquera, C., Simon, L., Martinez, A.,    Gutierrez Banos, J. L., et al. 2005. Fibroblast growth factor    receptor 3 is overexpressed in urinary tract carcinomas and    modulates the neoplastic cell growth. Clin Cancer Res 11:459-465.-   24. Tomlinson, D. C., Baldo, O., Harnden, P., and    Knowles, M. A. 2007. FGFR3 protein expression and its relationship    to mutation status and prognostic variables in bladder cancer. J    Pathol 213:91-98.-   25. van Rhijn, B. W., Montironi, R., Zwarthoff, E. C., Jobsis, A.    C., and van der Kwast, T. H. 2002. Frequent FGFR3 mutations in    urothelial papilloma. J Pathol 198:245-251.-   26. Tomlinson, D. C., Hurst, C. D., and Knowles, M. A. 2007.    Knockdown by shRNA identifies S249C mutant FGFR3 as a potential    therapeutic target in bladder cancer. Oncogene 26:5889-5899.-   27. Martinez-Torrecuadrada, J., Cifuentes, G., Lopez-Serra, P.,    Saenz, P., Martinez, A., and Casal, J. I. 2005. Targeting the    extracellular domain of fibroblast growth factor receptor 3 with    human single-chain Fv antibodies inhibits bladder carcinoma cell    line proliferation. Clin Cancer Res 11:6280-6290.-   28. Martinez-Torrecuadrada, J. L., Cheung, L. H., Lopez-Serra, P.,    Barderas, R., Canamero, M., Ferreiro, S., Rosenblum, M. G., and    Casal, J. I. 2008. Antitumor activity of fibroblast growth factor    receptor 3-specific immunotoxins in a xenograft mouse model of    bladder carcinoma is mediated by apoptosis. Mol Cancer Ther    7:862-873.-   29. Ornitz, D. M., and Leder, P. 1992. Ligand specificity and    heparin dependence of fibroblast growth factor receptors 1 and 3. J    Biol Chem 267:16305-16311.-   30. d'Avis, P. Y., Robertson, S. C., Meyer, A. N., Bardwell, W. M.,    Webster, M. K., and Donoghue, D. J. 1998. Constitutive activation of    fibroblast growth factor receptor 3 by mutations responsible for the    lethal skeletal dysplasia thanatophoric dysplasia type I. Cell    Growth Differ 9:71-78.-   31. Adar, R., Monsonego-Ornan, E., David, P., and Yayon, A. 2002.    Differential activation of cysteine-substitution mutants of    fibroblast growth factor receptor 3 is determined by cysteine    localization. J Bone Miner Res 17:860-868.-   32. Knowles, M. A. 2008. Novel therapeutic targets in bladder    cancer: mutation and expression of FGF receptors. Future Oncol    4:71-83.-   33. Naski, M. C., Wang, Q., Xu, J., and Ornitz, D. M. 1996. Graded    activation of fibroblast growth factor receptor 3 by mutations    causing achondroplasia and thanatophoric dysplasia. Nat Genet    13:233-237.-   34. Plotnikov, A. N., Schlessinger, J., Hubbard, S. R., and    Mohammadi, M. 1999. Structural basis for FGF receptor dimerization    and activation. Cell 98:641-650.-   35. Olsen, S. K., Ibrahimi, O. A., Raucci, A., Zhang, F.,    Eliseenkova, A. V., Yayon, A., Basilico, C., Linhardt, R. J.,    Schlessinger, J., and Mohammadi, M. 2004. Insights into the    molecular basis for fibroblast growth factor receptor autoinhibition    and ligand-binding promiscuity. Proc Natl Acad Sci USA 101:935-940.-   36. Jebar, A. H., Hurst, C. D., Tomlinson, D. C., Johnston, C.,    Taylor, C. F., and Knowles, M. A. 2005. FGFR3 and Ras gene mutations    are mutually exclusive genetic events in urothelial cell carcinoma.    Oncogene 24:5218-5225.-   37. Ellman, G. L. 1959. Tissue sulfhydryl groups. Arch Biochem    Biophys 82:70-77.-   38. Adams, G. P., and Weiner, L. M. 2005. Monoclonal antibody    therapy of cancer. Nat Biotechnol 23:1147-1157.-   39. Gong, Q., Ou, Q., Ye, S., Lee, W. P., Cornelius, J., Diehl, L.,    Lin, W. Y., Hu, Z., Lu, Y., Chen, Y., et al. 2005. Importance of    cellular microenvironment and circulatory dynamics in B cell    immunotherapy. J Immunol 174:817-826.-   40. Cappellen, D., De Oliveira, C., Ricol, D., de Medina, S.,    Bourdin, J., Sastre-Garau, X., Chopin, D., Thiery, J. P., and    Radvanyi, F. 1999. Frequent activating mutations of FGFR3 in human    bladder and cervix carcinomas. Nat Genet 23:18-20.-   41. Qiu, W. H., Zhou, B. S., Chu, P. G., Chen, W. G., Chung, C.,    Shih, J., Hwu, P., Yeh, C., Lopez, R., and Yen, Y. 2005.    Over-expression of fibroblast growth factor receptor 3 in human    hepatocellular carcinoma. World J Gastroenterol 11:5266-5272.-   42. Cortese, R., Hartmann, O., Berlin, K., and Eckhardt, F. 2008.    Correlative gene expression and DNA methylation profiling in lung    development nominate new biomarkers in lung cancer. Int J Biochem    Cell Biol 40:1494-1508.-   43. Woenckhaus, M., Klein-Hitpass, L., Grepmeier, U., Merk, J.,    Pfeifer, M., Wild, P., Bettstetter, M., Wuensch, P., Blaszyk, H.,    Hartmann, A., et al. 2006. Smoking and cancer-related gene    expression in bronchial epithelium and non-small-cell lung cancers.    J Pathol 210:192-204.-   44. Xin, X., Abrams, T. J., Hollenbach, P. W., Rendahl, K. G., Tang,    Y., Oei, Y. A., Embry, M. G., Swinarski, D. E., Garrett, E. N.,    Pryer, N. K., et al. 2006. CHIR-258 is efficacious in a newly    developed fibroblast growth factor receptor 3-expressing orthotopic    multiple myeloma model in mice. Clin Cancer Res 12:4908-4915.-   45. Davies, H., Bignell, G. R., Cox, C., Stephens, P., Edkins, S.,    Clegg, S., Teague, J., Woffendin, H., Garnett, M. J., Bottomley, W.,    et al. 2002. Mutations of the BRAF gene in human cancer. Nature    417:949-954.-   46. Trudel, S., Stewart, A. K., Rom, E., Wei, E., Li, Z. H., Kotzer,    S., Chumakov, I., Singer, Y., Chang, H., Liang, S. B., et al. 2006.    The inhibitory anti-FGFR3 antibody, PRO-001, is cytotoxic to t(4;14)    multiple myeloma cells. Blood 107:4039-4046.-   47. Gozlan, H., and Ben-Ari, Y. 1995. NMDA receptor redox sites: are    they targets for selective neuronal protection? Trends Pharmacol Sci    16:368-374.-   48. Hudziak, R. M., Lewis, G. D., Winget, M., Fendly, B. M.,    Shepard, H. M., and Ullrich, A. 1989. p185HER2 monoclonal antibody    has antiproliferative effects in vitro and sensitizes human breast    tumor cells to tumor necrosis factor. Mol Cell Biol 9:1165-1172.-   49. Masui, H., Kawamoto, T., Sato, J. D., Wolf, B., Sato, G., and    Mendelsohn, J. 1984. Growth inhibition of human tumor cells in    athymic mice by anti-epidermal growth factor receptor monoclonal    antibodies. Cancer Res 44:1002-1007.-   50. Pai, R., Dunlap, D., Qing, J., Mohtashemi, I., Hotzel, K., and    French, D. M. 2008. Inhibition of fibroblast growth factor 19    reduces tumor growth by modulating beta-catenin signaling. Cancer    Res 68:5086-5095.-   51. Pegram, M., Hsu, S., Lewis, G., Pietras, R., Beryt, M.,    Sliwkowski, M., Coombs, D., Baly, D., Kabbinavar, F., and    Slamon, D. 1999. Inhibitory effects of combinations of HER-2/neu    antibody and chemotherapeutic agents used for treatment of human    breast cancers. Oncogene 18:2241-2251.-   52. Lee, C. V., Liang, W. C., Dennis, M. S., Eigenbrot, C.,    Sidhu, S. S., and Fuh, G. 2004. High-affinity human antibodies from    phage-displayed synthetic Fab libraries with a single framework    scaffold. J Mol Biol 340:1073-1093.-   53. Liang, W. C., Dennis, M. S., Stawicki, S., Chanthery, Y., Pan,    Q., Chen, Y., Eigenbrot, C., Yin, J., Koch, A. W., Wu, X., et    al. 2007. Function blocking antibodies to neuropilin-1 generated    from a designed human synthetic antibody phage library. J Mol Biol    366:815-829.-   54. Carter, P., Presta, L., Gorman, C. M., Ridgway, J. B., Henner,    D., Wong, W. L., Rowland, A. M., Kotts, C., Carver, M. E., and    Shepard, H. M. 1992. Humanization of an anti-p185HER2 antibody for    human cancer therapy. Proc Natl Acad Sci USA 89:4285-4289.-   55. Sidhu, S. S., Li, B., Chen, Y., Fellouse, F. A., Eigenbrot, C.,    and Fuh, G. 2004. Phage-displayed antibody libraries of synthetic    heavy chain complementarity determining regions. J Mol Biol    338:299-310.-   56. Otwinowski, Z.a.M., W. 1997. Processing of X-ray diffraction    data collected in oscillation mode. Methods in Enzymology    276:307-326.-   57. McCoy, A. J., Grosse-Kunstleve, R. W., Storoni, L. C., and    Read, R. J. 2005. Likelihood-enhanced fast translation functions.    Acta Crystallogr D Biol Crystallogr 61:458-464.-   58. Emsley, P., and Cowtan, K. 2004. Coot: model-building tools for    molecular graphics. Acta Crystallogr D Biol Crystallogr    60:2126-2132.-   59. Murshudov, G. N., Vagin, A. A., and Dodson, E. J. 1997.    Refinement of macromolecular structures by the maximum-likelihood    method. Acta Crystallogr D Biol Crystallogr 53:240-255.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention.

1. An anti-FGFR3 antagonist antibody comprising: (i) a light chainvariable region comprising (a) HVR-L1 comprising the amino acid sequenceof SEQ ID NO:87, (b) HVR-L2 comprising the amino acid sequence of SEQ IDNO:88, and (c) HVR-L3 comprising the amino acid sequence of SEQ IDNO:89, and (ii) a heavy chain variable region comprising (a) HVR-H1comprising the amino acid sequence of SEQ ID NO:84, (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:85, and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:86, wherein saidantibody is conjugated to one or more cytotoxic agents.
 2. The antibodyof claim 1, wherein at least a portion of the framework sequence of theanti-FGFR3 antagonist antibody is a human consensus framework sequence.3. The antibody of claim 2, wherein the anti-FGFR3 antagonist antibodycomprises a human κ subgroup consensus framework sequence.
 4. Theantibody of claim 2, wherein the anti-FGFR3 antagonist antibodycomprises a heavy chain human subgroup III consensus framework sequence.5. The antibody of claim 1, wherein the anti-FGFR3 antagonist antibodycomprises an Fc domain.
 6. The antibody of claim 5, wherein the Fcdomain comprises a D265A/N297A (DANA) mutation.
 7. The antibody of claim1, wherein the anti-FGFR3 antagonist antibody comprises a light chainvariable region comprising the amino acid sequence set forth in SEQ IDNO:133.
 8. The antibody of claim 1, wherein the anti-FGFR3 antagonistantibody comprises a heavy chain variable region comprising the aminoacid sequence set forth in SEQ ID NO:132.
 9. The antibody of claim 1,wherein the anti-FGFR3 antagonist antibody is a monoclonal antibody. 10.The antibody of claim 1, wherein the anti-FGFR3 antagonist antibody isselected from the group consisting of a chimeric antibody, a humanizedantibody, an affinity matured antibody, a human antibody, and abispecific antibody.
 11. The method of claim 13, wherein said canceroverexpresses FGFR3.
 12. The method of claim 13, wherein said cancer isselected from the group consisting of bladder cancer and multiplemyeloma.
 13. A method of treating cancer in a subject in need thereofcomprising administering to the subject the antibody of claim
 1. 14-23.(canceled)
 24. The antibody of claim 1, wherein said cytotoxic agent isselected from the group consisting of a chemotherapeutic agent, a drug,a growth inhibitory agent, a toxin, and a radioactive isotope.